Identification and use of molecules implicated in pain

ABSTRACT

The invention relates to the use of:  
     Use of:  
     (a) an isolated gene sequence that is up-regulated in the spinal cord of a mammal in response to mechanistically distinct first and second models of neuropathic or central sensitization pain;  
     (b) an isolated gene sequence comprising a nucleic acid sequence of Table 1;  
     (c) an isolated gene sequence having at least 80% sequence identity with a nucleic acid sequence of Table 1;  
     (d) an isolated nucleic acid sequence that is hybridizable to any of the gene sequences according to (a), (b) or (c) under stringent hybridisation conditions;  
     (e) a recombinant vector comprising a gene sequence or nucleic acid sequence according to any one of (a) to (d);  
     (f) a host cell comprising the vector according to (e);  
     (g) a non-human animal having in its genome an introduced gene sequence or nucleic acid sequence or a removed or down-regulated gene sequence or nucleic acid sequence according to any one of (a) to (d);  
     (h) an isolated polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence encoded by a nucleotide sequence according to any one of (a) to (d), or a polypeptide variant thereof with sequential amino acid deletions from the C terminus and/or the N-terminus;  
     (i) an isolated polypeptide encoded by a nucleotide sequence according to any one of (a) to (d); or  
     (j) an isolated antibody that binds specifically to a polypeptide according to (h) or (i);  
     in the screening of compounds for the treatment of pain, or for the diagnosis of pain.  
     The invention also relates to the use of naturally occurring compounds such as peptide ligands of the expression products of the above gene sequences and their associated signal transduction pathways for use in the treatment of pain.

[0001] This application claims the priority of United Kingdomapplication NO. 0118354.0, filed Jul. 27, 2001, and United Kingdomapplication NO. 0202892.6, filed Feb. 7, 2002; the entire contents ofwhich applications are hereby incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to nucleic acids, their expressionproducts and pathways involved in pain, and their use in screening formolecules that can alleviate pain. The invention further relates tomethods for the assay and diagnosis of pain in patients.

BACKGROUND OF THE INVENTION

[0003] Pain is currently classified into four general types.Post-operative acute pain can be successfully treated with existing painmedications of e.g. the opioid and non-steroidal anti-inflammatory(NSAID) types, and is usually short-term and self-limiting. A secondtype of pain, present e.g. in cancer and arthritis, is also responsiveto medication initially with a NSAID and in its later stages withopioids. Neuropathic pain arises from damage to the central orperipheral nerve systems, and is more effectively treated withantidepressants or anticonvulsants. A fourth type of pain called centralsensitization results from changes in the central nervous system as aresult of chronic pain, these changes often being irreversible anddifficult to treat. Nerve pain from shingles or diabetes falls into thisand the neuropathic category. Changes occur where pain is at firstpoorly controlled and gradually progress to the point where a person issensitive to stimuli which would not normally cause pain, for example alight touch. People with pain of this kind often describe a widening ofthe pain area to include areas which had originally not been injured orwhich were thought not to be involved in pain. This classification is,however based on clinical symptoms rather than on the underlying painmechanisms.

[0004] Opiates such as morphine belong to a traditional class ofpain-relieving compounds that are now recognized as binding to opiatereceptors. Naturally occurring polypeptides have also been found to haveopiate-like effects on the central nervous system, and these includeβ-endorphin, met-enkephalin and leu-enkephalin.

[0005] Salicin was isolated at the beginning of the 19th century, andfrom that discovery a number of NSAIDs such as aspirin, paracetamol,ibuprofen, flurbiprofen and naproxen were developed. NSAIDs are by farthe most widely used pain-relieving compounds, but can exhibit sideeffects, in particular irritation of the GI tract that can lead to theformation of ulcers, gastrointestinal bleeding and anemia.

[0006] Interest in the neurobiology of pain is developing: see acolloquium sponsored by the US National Academy of Sciences in December1998 concerning the neurobiology of pain and reviewed in The Scientist13[1], 12, 1999. Many pain mechanisms were discussed including the roleof the capsaicin receptors in pain, (M. J. Caterina et al., Nature, 389,816-824, 1997). Large dosages of capsaicin were reported to disable thatreceptor, (W. R. Robbins et al., Anesthesia and Analgesia, 86, 579-583,1998). Additionally, a tetrodotoxin-resistant sodium channel found insmall diameter pain-sensing neurons (PN3) was discussed (A. N. Akopianet al., Nature, 379, 257-262, 1986) and L. Sangameswaran et al., Journalof Biological Chemistry, 271, 953-956, 1996). Its involvement intransmission and sensitization to pain signals has been reported, (S. D.Novakovic et al., Journal of Neuroscience, 18, 2174-2187, 1998). Afurther tetrodotoxin-resistant sodium channel has been reported (S. Tateet al., Nature Neuroscience, 1, 653-655 1998).

[0007] Second messenger systems have also been shown to be importantsince knockout-mice lacking protein kinase C (PKC) γ were reported torespond to acute pain e.g. from a hot surface, but not to respond toneuropathic pain when their spinal nerves are injured (Malmberg et al.,Science, 278, 279-283 (1997).

[0008] Present methods for identifying novel compounds that relieve painof one or more of the types indicated above suffer from the defect thatthey are dependent either on the relatively limited number of receptorsknown to be involved in pain or on the empirical identification of newreceptors which is an uncertain process. In relation to known receptors,for example the opioid receptor, research directed to improved compoundsoffers the possibility of screening compounds that have a bettertherapeutic ratio and fewer side effects. This does not lead naturallyto compounds for different pain receptors that have new modes of actionand new and qualitatively different benefits. Even when newly identifiedadditional receptors are taken into account, known receptors revolvearound tens of gene products. However, there are between 30,000 and40,000 genes in the genome of an animal and more of them are concernedwith nervous system function than with peripheral function. We thereforeconcluded that a large number of receptors and pathways are important tothe transduction of pain, but up to now have remained unknown.

SUMMARY OF THE INVENTION

[0009] It is an object of the invention to provide sequences of geneticmaterial for which no role in pain has previously been disclosed, andwhich are useful, for example, in:

[0010] identifying metabolic pathways for the transduction of pain

[0011] identifying from said metabolic pathways compounds having utilityin the diagnosis or treatment of pain

[0012] producing proteins and polypeptides with a role in thetransduction of pain;

[0013] producing genetically modified non-human animals that are usefulin the screening of compounds having utitlity in the treatment ordiagnosis of pain.

[0014] Identifying ligand molecules for receptors involved in saidmetabolic pathways and having utility in the treatment of pain.

[0015] It is yet a further object of the invention to provide researchtools, for example non-human animals and microorganisms, that can beused in screening compounds for pharmacological activity, especiallypain-reducing activity.

[0016] The present invention is based on sequences that are up-regulatedin two models of chronic pain, namely streptozocin-induced diabetes andchronic constrictive injury (CCI) to a nerve leading to the spine, forexample the sciatic nerve.

[0017] In one aspect, the invention relates to the use in the screeningof compounds that are effective in the treatment of pain, or in thediagnosis of pain, of:

[0018] (a) an isolated gene sequence that is up-regulated in the spinalcord of a mamal in response to first and second models of pain, forexample in response to streptozocin-induced diabetes and in response toa chronic constrictive injury to a nerve leading into the spine;

[0019] (b) an isolated gene sequence having at least 80% sequenceidentity with any of the the nucleic acid sequences of the accompanyingTable I in the specification, preferably 85% sequence identity, morepreferably 90%, increasingly preferably 95%, most preferably 99%.

[0020] (c) an isolated nucleic acid comprising a sequence that ishybridizable to any of the gene sequences according to (a) or (b) understringent hybridisation conditions;

[0021] (d) a recombinant vector comprising any one of the gene sequencesaccording to (a) to (c);

[0022] (e) a host cell containing the vector according to (d);

[0023] (f) a non-human animal, for use in the screening of compoundsthat are effective in the treatment of pain, or in the diagnosis ofpain, having in its genome an introduced gene sequence or a removed ordown-regulated nucleotide sequence, said sequence becoming up-regulatedin the spinal cord of a mammal in response to first and second models ofpain, particularly neuropathic or sensitisation pain, for example inresponse to streptozocin-induced diabetes and in response to a chronicconstrictive injury to a nerve leading into the spine;

[0024] (g) an isolated polypeptide containing an amino acid sequence atleast 90% identical to an amino acid sequence encoded by a nucleotidesequence according to any one of (a) to (d), or a variant thereof withsequential amino acid deletions from the C terminus and/or theN-terminus; or

[0025] (h) an isolated antibody that binds to the isolated polypeptideaccording to (g).

[0026] The invention further provides a compound that is useful in thetreatment or diagnosis of pain and that modulates the action of anexpression product of a gene sequence that becomes up-regulated in thespinal cord of a mammal in response to first and second models of pain,for example being up-regulated both in response to streptozocin induceddiabetes and in response to chronic constrictive injury to a nerveleading into the spine.

[0027] The invention also relates to the use of naturally occurringcompounds such as peptide ligands of the expression products of theabove gene sequences and their associated signal transduction pathwaysfor the treatment of pain.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0028] Definitions

[0029] Within the context of the present invention:

[0030] “Comprising” encompasses consisting of and including. Thusnucleic acid comprising a defined sequence includes nucleic acid thatmay contain a full-length gene or full-length cDNA. The gene may includeany of the naturally occurring regulatory sequence(s), such as atranscription and translation start site, a promoter, a TATA box in thecase of eukaryotes, and transcriptional and translational stop sites.Further, a nucleic acid sequence comprising a cDNA or gene may includeany appropriate regulatory sequences for the efficient expressionthereof in vitro.

[0031] “Isolated” requires that the material be removed from itsoriginal environment (e.g. the natural environment if it is naturallyoccurring). For example, a naturally occurring polynucleotide or apeptide present in a living animal is not isolated, but the samepolynucleotide or peptide, separated from some or all of the coexistingmaterials in the natural system, is isolated. Such polynucleotide can bepart of a vector and/or such polynucleotide or peptide can be part of acomposition, and still be isolated in that the vector or composition isnot a part of its natural environment.

[0032] “Mechanistically distinct” in relation to pain models impliesthat the pain is induced by mechanisms that differ in kind rather thanbeing variants of a similar pain model. Thus diabetic pain and chronicconstrictive pain models are mechanistically distinct whereas spinalnerve ligation models and sciatic nerve ligation models are not.

[0033] “Purified” does not require absolute purity; instead it isintended as a relative definition. Purification of starting materials ornatural materials from their native environment to at least one order ofmagnitude, preferably two or three orders, and more preferably four orfive orders of magnitude is expressly contemplated.

[0034] “Nucleic acid sequence” or “gene sequence” means a sequence ofnucleotides or any variant or homologue thereof, or truncated orextended sequence thereof, and is preferably indicated by a Genebankaccession number. Also within the scope of the present invention areup-regulated nucleic acid sequences which encode expression productswhich are components of signaling pathways. This invention also includesany variant or homologue or truncated or extended sequence of theup-regulated nucleotide sequence. Also within the scope of the presentinvention, the term “nucleic acid(s) product”, or “expression product”or “gene product” or a combination of these terms refers without beingbiased, to any, protein(s), polypeptide(s), peptide(s) or fragment(s)encoded by the up-regulated nucleotide sequence.

[0035] “Operably linked” refers to a linkage of polynucleotide elementsin a functional relationship. For instance, a promoter or an enhancer isoperably linked to a coding sequence if it regulates the transcriptionof the coding sequence. In particular, two DNA molecules (such as apolynucleotide containing a promoter region and a polynucleotideencoding a desired polypeptide) are said to be “operably linked” if thenature of the linkage between the two polynucleotides does not (1)result in the introduction of a frame-shift mutation and (2) interferewith the ability of the polynucleotide containing the promoter to directthe transcription of the coding polynucleotide.

[0036] “Gene product” refers to polypeptide—which is interchangeablewith the term protein—which is encoded by a nucleotide sequence andincludes single-chain polypeptide molecules as well asmultiple-polypeptide complexes where individual constituent polypeptidesare linked by covalent or non-covalent means.

[0037] Polypeptides of the present invention may be produced bysynthetic means (e.g. as described by Geysen et al., 1996) or byrecombinant means.

[0038] The terms “variant”, “homologue”, “fragment”, “analogue” or“derivative” in relation to the amino acid sequence for the polypeptideof the present invention includes any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) amino acid from or to the sequence providing the resultantpolypeptide has the native gene product activity. In particular, theterm “homologue” covers homology with respect to structure and/orfunction. With respect to sequence homology, there is at least 90%, morepreferably at least 95% homology to an amino acid sequence encoded bythe relevant nucleotide sequence shown in Table 1, preferably there isat least 98% homology.

[0039] Typically, for the variant, homologue or fragment of the presentinvention, the types of amino acid substitutions that could be madeshould maintain the hydrophobicity/hydrophilicity of the amino acidsequence. Amino acid substitutions may include the use of non-naturallyoccurring amino acid analogues.

[0040] In addition, or in the alternative, the protein itself could beproduced using chemical methods to synthesize a polypeptide, in whole orin part. For example, peptides can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography (e.g. Creighton (1983) ProteinsStructures and Molecular Principles, W H Freeman and Co., New York,N.Y., USA). The composition of the synthetic peptides may be confirmedby amino acid analysis or sequencing (e.g. the Edman degradationprocedure).

[0041] Direct peptide synthesis can be performed using varioussolid-phase techniques (Roberge J Y et al Science Vol 269 1995 202-204)and automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the instructionsprovided by the manufacturer. Additionally, the amino acid sequence of agene product, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with a sequence fromother subunits, or any part thereof, to produce a variant polypeptide.

[0042] In another embodiment of the invention, a gene product natural,modified or recombinant amino acid sequence may be ligated to aheterologous sequence to encode a fusion protein. For example, forscreening of libraries for compounds and peptide agonists andantagonists of gene product activity, it may be useful to encode achimeric gene product expressing a heterologous epitope that isrecognised by a commercially available antibody. A fusion protein mayalso be engineered to contain a cleavage site located between a geneproduct sequence and the heterologous protein sequence, so that the geneproduct may be cleaved and purified away from the heterologous moiety.

[0043] The gene product may also be expressed as a recombinant proteinwith one or more additional polypeptide domains added to facilitateprotein purification. Such purification facilitating domains include,but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilisedmetals (Porath J, Protein Expr Purif Vol 3 1992 p 263-281), protein Adomains that allow purification on immobilised immunoglobulin, and thedomain utilised in the FLAGS extension/affinity purification system(Immunex Corp, Seattle, Wash., USA). The inclusion of a cleavable linkersequence such as Factor XA or enterokinase (Invitrogen, San Diego,Calif., USA) between the purification domain and the gene product isuseful to facilitate purification.

[0044] “Pain” includes chronic pain and in particular diabetic pain.

[0045] “Stringent hybridization conditions” is a recognized term in theart and for a given nucleic acid sequence refers to those conditionswhich permit hybridization of that sequence to its complementarysequence and closely homologous nucleic acid sequences. Conditions ofhigh stringency may be illustrated in relation to filter-bound DNA asfor example 2×SCC, 65° C. (where SSC=0.15M sodium chloride, 0.015Msodium citrate, pH 7.2); or as 0.5M NaHPO₄, 7% sodium dodecyl sulfate(SDS), 1 mM EDTA, at 65° C., and washing in 0.1×SCC/0.1% SDS at 68° C.(Ausubel F. M. et al., eds, 1989, Current Protocols in MolecularBiology, Vol. I, Green Publishing Associates, Inc., and John Wiley &Sons Inc., New York, at p. 2. 10.3). Hybridization conditions can berendered highly stringent by raising the temperature and/or by theaddition of increasing amounts of formamide, to destabilize the hybridduplex of non-homologous nucleic acid sequence relative to homologousand closely homologous nucleic acid sequences. Thus, particularhybridisation conditions can be readily manipulated, and will generallybe chosen depending on the desired results.

[0046] “Variants or homologues” include (a) sequence variations ofnaturally existing gene(s) resulting from polymorphism(s), mutation(s),or other alteration(s) as compared to the above identified sequences,and which do not deprive the encoded protein of function (b) recombinantDNA molecules, such as cDNA molecules encoding genes indicated by therelevant Genebank accession numbers and (c) any sequence that hybridizeswith the above nucleic acids under stringent conditions and encodes afunctional protein or fragment thereof.

[0047] Identified Sequences

[0048] The inventors have identified nucleotide sequences that give riseto expression products listed in Table 1, that become differentiallyexpressed in the spinal cord in response to two distinct chronic painstimuli, for example neuropathic pain stimuli, and that are believed tobe involved in the transduction of pain. In Table 1, * denotes morepreferred nucleotide sequences and ** denotes most preferred nucleotidesequences. These nucleotide sequences have not previously beenimplicated in the transduction of pain.

[0049] The validity of the present experimental procedure was confirmedby the fact that nucleotide sequences were obtained as a result of theinvestigation whose function in the transduction of pain has beenpreviously confirmed and established. These nucleic acid sequences arenot part of this invention. Any of the nucleic acid sequences andexpression products can be used to develop screening technologies forthe identification of novel molecules for the prevention or treatment ofpain. These screening technologies could also be used to ascribe newpain therapeutic indications to molecules that have not previously beenidentified as being useful for the prevention or treatment of pain.Furthermore, the said nucleic acid sequences can be used as diagnostictools and for the development of diagnostic tools.

[0050] Production of Polypeptides and Nucleic Acids

[0051] Vectors

[0052] Recombinant expression vectors comprising a nucleic acid can beemployed to express any of the nucleic acid sequences of the invention.The expression products derived from such vector constructs can be usedto develop screening technologies for the identification of moleculesthat can be used to prevent or treat pain, and in the development ofdiagnostic tool for the identification and characterization of pain. Theexpression vectors may also be used for constructing transgenicnon-human animals.

[0053] Gene expression requires that appropriate signals be provided inthe vectors, said signals including various regulatory elements such asenhancers/promoters from viral and/or mammalian sources that driveexpression of the genes or nucleotide sequences of interest in hostcells. The regulatory sequences of the expression vectors used in theinvention are operably linked to the nucleic acid sequence encoding thepain-associated protein of interest or a peptide fragment thereof.

[0054] Generally, recombinant expression vectors include origins ofreplication, selectable markers, and a promoter derived from a highlyexpressed gene to direct transcription of a downstream nucleotidesequence. The nucleotide sequence is assembled in an appropriate framewith the translation, initiation and termination sequences, and ifapplicable a leader sequence to direct the expression product to theperiplasmic space, the extra-cellular medium or cell membrane.

[0055] In a specific embodiment wherein the vector is adapted forexpressing desired sequences in mammalian host cells, the preferredvector will comprise an origin of replication from the desired host, asuitable promoter and an enhancer, and also any necessary ribosomebinding sites, polyadenylation site, transcriptional terminationsequences, and optionally 5′-flanking non-transcribed sequences. DNAsequences derived from the SV40 or CMV viral genomes, for example SV40or CMV origin, early promoters, enhancers, and polyadenylation sites maybe used to provide the required non-transcribed genetic elements.

[0056] A recombinant expression vector used in the inventionadvantageously also comprises an untranscribed polynucleotide regionlocated at the 3′end of the coding sequence open reading frame (ORF),this 3′-untranslated region (UTR) polynucleotide being useful forstabilizing the corresponding mRNA or for increasing the expression rateof the vector insert if this 3′-UTR harbours regulation signal elementssuch as enhancer sequences.

[0057] Suitable promoter regions used in the expression vectors arechosen taking into account the host cell in which the nucleic acidsequence is to be expressed. A suitable promoter may be heterologouswith respect to the nucleic acid sequence for which it controls theexpression, or alternatively can be endogenous to the nativepolynucleotide containing the coding sequence to be expressed.Additionally, the promoter is generally heterologous with respect to therecombinant vector sequences within which the construct promoter/codingsequence has been inserted. Preferred promoters are the LacI, LacZ, T3or T7 bacteriophage RNA polymerase promoters, the lambda P_(R), P_(L)and Trp promoters (EP-0 036 776), the polyhedrin promoter, or the p10protein promoter from baculovirus (kit Novagen; Smith et al., (1983);O'Reilly et al. (1992).

[0058] Preferred selectable marker genes contained in the expressionrecombinant vectors used in the invention for selection of transformedhost cells are preferably dehydrofolate reductase or neomycin resistancefor eukaryotic cell culture, TRP1 for S. cerevisiae or tetracycline,rifampicin or ampicillin resistance in E. coli, or Levamsaccharase forMycobacteria, this latter marker being a negative selection marker.

[0059] Preferred bacterial vectors are listed hereafter as illustrativebut not limitative examples: pQE70, pQE60, pQE-9 (Quiagen), pD10,phagescript, psiX174, p.Bluescript SK, pNH8A, pNH16A, pNH18A, pNH46A(Stratagene); pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO,pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL(Pharmacia); pQE-30 (QIA express).

[0060] Preferred bacteriophage recombinant vectors of the invention areP1 bacteriophage vectors such as described by Sternberg N. L. (1992;1994).

[0061] A suitable vector for the expression of any of the painassociated polypeptides used in the invention or fragments thereof, is abaculovirus vector that can be propagated in insect cells and in insectcell-lines. A specific suitable host vector system is the pVL 1392/1393baculovirus transfer vector (Pharmingen) that is used to transfect theSF9 cell line (ATCC N°CRL 1711) that is derived from Spodopterafrugiperda.

[0062] The recombinant expression vectors of the invention may also bederived from an adenovirus. Suitable adenoviruses are described byFeldman and Steig (1996) or Ohno et al. (1994). Another preferredrecombinant adenovirus is the human adenovirus type two or five (Ad 2 orAd 5) or an adenovirus of animal origin (Patent Application WO94/26914).

[0063] Particularly preferred retrovirus for the preparation orconstruction of retroviral in vitro or in vivo gene delivery vehiclesinclude retroviruses selected from the group consisting of Mink-CellFocus Inducing Virus, Murine Sarcoma Virus, and Ross Sarcoma Virus.Other preferred retroviral vectors are those described in Roth et al.(1996), in PCT Application WO 93/25234, in PCT Application WO 94/06920,and also in Roux et al. (1989), Julan et al. (1992) and Nada et al.(1991).

[0064] Yet, another viral vector system that is contemplated is theAdeno Associated Viruses (AAV) such as those described by Flotte et al.(1992), Samulski et al. (1989) and McLaughlin et al. (1996).

[0065] Host cells Expressing Pain Associated Polypeptides

[0066] Host cells that endogenously express pain associated polypeptidesor have been transformed or transfected with one of the nucleic acidsequences described herein, or with one of the recombinant vectordescribed above, particularly a recombinant expression vector, can beused in the present invention. Also included are host cells that aretransformed (prokaryotic cells) or are transfected (eukaryotic cells)with a recombinant vector such as one of those described above.

[0067] Preferred host cells used as recipients for the expressionvectors used in the invention are the following:

[0068] (a) prokaryotic host cells: Escherichia coli, strains. (i.e.DH5-α, strain) Bacillus subtilis, Salmonella typhimurium and strainsfrom species like Pseudomonas, Streptomyces and Staphylococcus for theexpression of up and down-regulated nucleic acid sequences modulated bypain, characterized by having at least 80% sequence identity with any ofthe nucleic acid sequences of Table 1. Plasmid propagation in these hostcells can provide plasmids for transfecting other cells.

[0069] (b) eukaryotic host cells: HeLa cells (ATCC N°CCL2; N°CCL2.1;N°CCL2.2), Cv 1 cells (ATCC N°CCL70), COS cells (ATCC N°CRL 1650; N°CRL1651), Sf-9 cells (ATCC N°CRL 1711), C127 cells (ATCC N°CRL-1804), 3T3cells (ATCC N°CRL-6361), CHO cells (ATCC N°CCL-61), human kidney 293cells (ATCC N° 45504; N°CRL-1573), BHK (ECACC N°84100 501; N°84111301),PC12 (ATCC N°CRL-1721), NT2, SHSY5Y (ATCC N° CRL-2266), NG108 (ECACCN°88112302) and F11, SK-N-SH (ATCC N° CRL-HTB-11), SK-N-BE (2) (ATCCN°CRL-2271), IMR-32 (ATCC N°CCL-127). A preferred system to which thenucleic acids of the invention can be expressed are neuronal cell linessuch as PC 12, NT2, SHSYSY, NG108 and Fl 1, SK-N-SH, SK-N-BE (2), IMR-32cell lines, COS cells, 3T3 cells, HeLa cells, 292 cells and CHO cells.The above cell lines could be used for the expression of any of thenucleic acid sequences of Table 1.

[0070] When a nucleic acid sequence of the Table 1 is expressed using aneuronal cell line, the sequence can be expressed under an endogenouspromoter or native neuronal promoter or an exogenous promoter. Suitableexogenous promoters include SV40 and CMV and eukaryotic promoters suchas the tetracycline promoter. The preferred promoter when painassociated molecules are endogenously expressed is an endogenouspromoter. A preferred promoter in a recombinant cell line is the CMVpromoter.

[0071] In a specific embodiment of the host cells described above, thesehost cells have also been transfected or transformed with apolynucleotide or a recombinant vector for the expression of a naturalligand of any of the nucleic acid sequences of Table 1 or a modulator ofthese expression products.

[0072] Proteins, Polypeptides and Fragments

[0073] The expression products of the nucleic acid sequences of Table Ibelow or fragment(s) thereof can be prepared using recombinanttechnology, from cell lines or by chemical synthesis. Recombinantmethods, chemical methods or chemical synthetic methods can be used tomodify a gene in order to introduce into the gene product, or a fragmentof the gene product, features such as recognition tags, cleavage sitesor other modifications. For efficient polypeptide production, theendogenous expression system or recombinant expression system shouldallow the expression products to be expressed in a manner that willallow the production of a functional protein or fragment thereof whichcan be purified. Preferred cell lines are those that allow high levelsof expression of polypeptide or fragments thereof. Such cell linesinclude cell lines which naturally express any of the nucleic acidsequences of Table 1 or common mammalian cell lines such as CHO cells orCOS cells, etc, or more specific neuronal cell lines such as PC12.However, other cell types that are commonly used for recombinant proteinproduction such as insect cells, amphibian cells such as oocytes, yeastand prokaryotic cell lines such as E. coli can also be used.

[0074] The expression products of Table 1 or fragments thereof can beutilized in screens to identify potential therapeutic ligands, either asa purified protein, as a protein chimera such as those produced by phagedisplay, as a cell membrane (lipid or detergent) preparation, or inintact cells.

[0075] The invention also relates generally to the use of proteins,peptides and peptide fragments for the development of screeningtechnologies for the identification of molecules for the prevention ortreatment of pain, and the development of diagnostic tools for theidentification and characterization of pain. These peptides includeexpression products of the nucleic acid sequences of Table 1 andpurified or isolated polypeptides or fragments thereof having at least90%, preferably 95%, more preferably 98% and most preferably 99%sequence identity with the any of the expression products of nucleicacid sequences of Table 1. Expressed peptides and fragments of any ofthese nucleic acid sequences can be used to develop screeningtechnologies for the identification of novel molecules for theprevention or treatment of pain. These screening technologies could alsobe used to ascribe new pain therapeutic indications to molecules, whichhave not previously been ascribed for the prevention or treatment ofpain. Furthermore the said expressed peptides and fragments can be usedas diagnostic tools and for the development of diagnostic tools.

[0076] Screening Methods

[0077] As discussed above, we have identified nucleic acid sequenceswhose expression is regulated by pain, particularly chronic pain andmore particularly diabetic pain. The expression products of thesenucleic acids can be used for screening ligand molecules for theirability to prevent or treat pain, and particularly, but not exclusively,chronic pain. The main types of screens that can be used are describedbelow. The test compound can be a peptide, protein or chemical entity,either alone or in combination(s), or in a mixture with any substance.The test compound may even represent a library of compounds.

[0078] The expression products of any of the nucleic acid sequences ofTable 1 or fragments thereof can be utilized in a ligand binding screenformat, a functional screen format or in vivo format. Examples ofscreening formats are provided.

[0079] A) Ligand Binding Screen

[0080] In ligand binding screening a test compound is contacted with anexpression product of one of the sequences of Table 1, and the abilityof said test compound to interact with said expression product isdetermined, e.g. the ability of the test compound(s) to bind to theexpression product is determined. The expression product can be a partof an intact cell, lipidic preparation or a purified polypeptide(s),optionally attached to a support, such as beads, a column or a plateetc.

[0081] Binding of the test compound is preferably performed in thepresence of a ligand to allow an assessment of the binding activity ofeach test compound. The ligand may be contacted with the expressionproduct either before, simultaneously or after the test compound. Theligand should be detectable and/or quantifiable. To achieve this, theligand can be labeled in a number of ways, for example with achromophore, radioactive, fluorescent, phosphorescent, enzymatic orantibody label. Methods of labeling are known to those in the art. Ifthe ligand is not directly detectable it should be amenable to detectionand quantification by secondary detection, which may employ the abovetechnologies. Alternatively the expression product or fragment thereofcan be detectable or quantifiable. This can be achieved in a similarmanner to that described above.

[0082] Binding of the test compound modifies the interaction of theligand with its binding site and changes the affinity or binding of theligand for/to its binding site. The difference between the observedamount of ligand bound relative to the theoretical maximum amount ofligand bound (or to the ligand bound in the absence of a test compoundunder the same conditions) is a reflection of the binding ability (andoptionally the amount and/or affinity) of a test compound to bind theexpression product.

[0083] Alternatively, the amount of test compound bound to theexpression product can be determined by a combination of chromatographyand spectroscopy. This can also be achieved with technologies such asBiacore (Amersham Pharmacia). The amount of test compound bound to theexpression product can also be determined by direct measurement of thechange in mass upon compound or ligand binding to the expressionproduct. Alternatively, the expression product, compound or ligand canbe fluorescently labelled and the association of expression product withthe test compound can be followed by changes in Fluorescence EnergyTransfer (FRET).

[0084] The invention therefore includes a method of screening for painalleviating compounds, comprising:

[0085] a) contacting a test compound or test compounds in the presenceof a ligand with an expression product of any of the nucleic acidsequences of Table 1 or with cell expressing at least one copy of theexpression product or with a lipidic matrix containing at least one copyof the expression product; determining the binding of the test compoundto the expression product, and

[0086] b) selecting test compounds on the basis of their bindingabilities.

[0087] In the above method, the ligand may be added prior to,simultaneously with or after contact of the test compound with theexpression product. Non limiting examples and methodology can be gainedfrom the teachings of the Molecular Probes handbook and referencestherein (Molecular Probes, Inc., 4849 Pitchford Ave, Eugene, USA),Methods in neurotransmitter receptor analysis (Yamamura H I., Enna, SJ., and Kuhar, M J., Raven Press New York, the Glaxo Pocket Guide toPharmacology, Dr. Michael Sheehan, Glaxo Group Research Ltd, Ware, HertsSG12 ODP), Bylund D B and Murrin L C (2000, Life Sciences, 67 (24)2897-911), Owicki J C (2000, J. biomol Screen (5) 297-306), Alberts etal (1994, Molecular Biology of the Cell, 3^(rd) Edn, Garland PublicationInc), Butler J E, (2000 Methods 22(1):4-23, Sanberg S A (2000, Curr OpinBiotechnol 11(1) 47-53), and Christopoulos A (1999, biochem Pharmacol58(5) 735-48).

[0088] Functional Screening

[0089] (a) Kinase Assays

[0090] The expression products of any of the nucleic acid sequences ofTable 1 which encode a kinase are amenable to screening using kinaseassay technology.

[0091] Kinases have the ability to add phosphate molecules to specificresidues in ligands such as binding peptides in the presence of asubstrate such as adenosine triphosphate (ATP). Formation of a complexbetween the kinase, the ligand and substrate results in the transfer ofa phosphate group from the substrate to the ligand. Compounds thatmodulate the activity of the kinase can be determined with a kinasefunctional screen. Functional screening for modulators of kinaseactivity therefore involves contacting one or more test compounds withan expression product of one of the nucleic acid sequences of Table 1which encodes a kinase, and determining the ability of said testcompound to modulate the transfer of a phosphate group from thesubstrate to the ligand.

[0092] The expression product can be part of an intact cell or of alipidic preparation or it can be a purified polypeptide(s), optionallyattached to a support, for example beads, a column, or a plate. Bindingis preferably performed in the presence of ligand and substrate to allowan assessment of the binding activity of each test compound.

[0093] The ligand should contain a specific kinase recognition sequenceand it should not be phosphorylated at its phosphoryation site. Theligand and/or substrate may be contacted with the kinase either before,simultaneously or after the test compound. Optionally the substrate maybe labelled with a kinase transferable labelled phosphate. The assay ismonitored by the phosphorylation state of the substrate and/or theligand. The ligand should be such that its phosphorylation state can bedetermined. An alternative method to do this is to label the ligand witha phosphorylation-state-sensitive molecule. To achieve this, the ligandcan be labelled in a number of ways, for example with a chromophore,radioactive, fluorescent, phosphorescent, enzymatic or antibody label.If the ligand is not directly detectable it should be amenable todetection and quantification by secondary detection, which may employthe above technologies. Such technologies are known to those in the art.

[0094] Binding of the test compound to the kinase modifies its abilityto transfer a phosphate group from the substrate to the ligand. Thedifference between the observed amount of phosphate transfer relative tothe theoretical maximum amount of phosphate transfer is a reflection ofthe modulatory effect of the test compound. Alternatively, the degree ofphosphate transfer can be determined by a combination of chromatographyand spectroscopy. The extent of phosphorylation of the ligand peptide ordephosphorylation of the substrate can also be determined by directmeasurement. This can be achieved with technologies such as Biacore(Amersham Pharmacia).

[0095] The invention also provides a method for screening compounds forthe ability to relieve pain, which comprises:

[0096] (a) contacting one or more test compounds in the presence ofligand and substrate with an expression product of any of the nucleotidesequences of Table I which is a kinase or with a cell containing atleast 1 copy of the expression product or with a lipidic matrixcontaining at least 1 copy of an expression product;

[0097] (b) determining the amount of phosphate transfer from thesubstrate to the ligand; and

[0098] (c) selecting test compounds on the basis of their capacity tomodulate phosphate transfer.

[0099] Optionally ligand, substrate and/or other essential molecules maybe added prior to contacting the test compound with expression productof step (a) or after step (a). Non limiting examples and methodology canbe gained from the teachings of the Molecular Probes handbook andreferences therein (Molecular Probes, Inc., 4849 Pitchford Ave, Eugene,USA), Methods in Molecular Biology 2000; 99: 191-201, Oncogene 2000 20;19(49): 5690-701, and FASAB Journal, (10, 6, P55, P1458, 1996, Pocius DAmrein K et al).

[0100] b) Phosphatase Assays

[0101] The expression products of any of the nucleic acid sequences ofTable 1 which encode a phosphatase are amenable to screening usingphosphatase assay technology.

[0102] Phosphatase enzymes have the ability to remove phosphatemolecules from specific residues in ligands such as peptides. Thisreaction takes place in the presence of a substrate such as AdenosineDiphosphate (ADP). The complexing of the phosphatase polypeptide, ligandand substrate results in the transfer of a phosphate group from theligand to substrate. Compounds that modulate the activity of thePhosphatase can be determined with a Phosphatase functional screen. Thisscreen detects the reverse of the Kinase functional screen outlinedabove.

[0103] The invention also provides a method for screening compounds forthe ability to relieve pain, which comprises:

[0104] (a) contacting at one or more test compounds in the presence ofligand and substrate with an expression product of any of the nucleotidesequences of Table 1 which is a phosphatase or with a cell containing atleast 1 copy of the expression product or with a lipidic matrixcontaining at least 1 copy of an expression product;

[0105] (b) determining the amount of phosphate transfer from the ligandto the substrate; and

[0106] (c) selecting test compounds on the basis of their capacity tomodulate phosphate transfer.

[0107] Optionally ligand, substrate and/or other essential molecules maybe added prior to contacting the test compound with expression productof step (a) or after step (a). Non-limiting examples and methodology canbe gained from the teachings of the Molecular Probes handbook andreferences therein (Molecular Probes, Inc., 4849 Pitchford Ave, Eugene,USA), and FASAB Journal, (10, 6, P55, P1458, 1996, Pocius D Amrein K etal).

[0108] c) Phosphodiesterase Assays

[0109] An expression product of any of the nucleic acid sequences ofTable I which encodes a phosphodiesterase is amenable to screening usingphosphodiesterase assay technology.

[0110] Phosphodiesterases have the ability to cleave cyclic nucleotidescAMP (cyclic adenosine monophosphate) and/or cGMP (cyclic guanosinemonophosphate) (substrate) at their 3′phosphatase bond to form 5′AMP and5′GMP. Functional screening for modulators of phosphodiesterasepolypeptide comprises contacting one or more test compounds with anexpression product as set out above which is a phosphodiesterase anddetermining the ability of said test compound(s) to modulate thecleavage of cyclic nucleotides cAMP and/or cGMP at their 3′phosphatasebond to form 5′AMP and 5′GMP. The expression product can be part of anintact cell or lipidic preparation or a purified polypeptide(s),optionally attached to a support, for example beads, a column, or aplate. Binding is preferably performed in the presence of cAMP or cGMPto allow an assessment of the binding activity of each test compound.The cAMP or cGMP and other essential molecules may be contacted with thephosphodiesterase peptide either before, simultaneously or after thetest compound(s).

[0111] A characteristic of the cAMP or cGMP is that it can readily beradio labeled (Thompson et al, Advances in cyclic nucleotide research,10, 69-92 (1974)). The conversion of cAMP or cGMP to 5′AMP or 5′GMP canbe detected with the use of chromatography and separation technologies.Such technology is known to those in the art. Binding of the testcompound to the phosphodiesterase polypeptide modifies its ability toconvert cAMP or cGMP to 5′AMP or 5′GMP. The difference between theobserved amount of conversion relative to the theoretical maximum amountof conversion is a reflection of the modulatory effect of the testcompound(s).

[0112] A non-limiting general understanding of how to assay for theactivity of Phosphodiesterases can be gained from Horton, J K. AndBaxendale, P M (Methods in molecular Biology, 1995, 41, p 91-105, Eds.Kendall, D A. and Hill, S J, Humana Press, Towota, N.J.) and MolecularProbes handbook and references therein (Molecular Probes, Inc., 4849Pitchford Ave, Eugene, USA).

[0113] The invention also provides methods of screening for painalleviating compounds, comprising;

[0114] a) contacting one or more test compounds in the presence of cAMPand/or cGMP with an expression product of any of the nucleotidesequences of Table 1 which is a phosphodiesterase or with a cellexpressing at least 1 copy of an expression product or with a lipidicmatrix containing at least 1 copy of an expression product;

[0115] b) determining the amount of cAMP and/or cGMP converted to 5′AMPand/or 5′GMP, and

[0116] c) selecting test compounds on the basis of their ability tomodulate said conversion.

[0117] d) Ion Channel Protein Assays

[0118] An expression product of any of the nucleic acid sequences ofTable 1 which encodes an ion channel protein, and in particular any ofthe nucleic acid sequences listed in Table 1, is amenable to screeningusing ion channel protein assay technology.

[0119] Ion channels are membrane associated proteins. They are dividedinto three main groups:

[0120] (1) ligand gated ion channels;

[0121] (2) voltage gated ion channels; and

[0122] (3) mechano gated ion channels.

[0123] Ion channels allow the passage of ions through cellular membranesupon stimulation by ligand, change in membrane potential or physicalchanges in environment such as temperature and pH. Compounds thatmodulate the activity of ion channels can be determined with an ionchannel functional screen.

[0124] Functional screening for modulators of ion channels involvescontacting a test compound with an expression product as aforesaid whichis an ion channel protein or a fragment thereof and determining theability of said test compound to modulate the activity of saidexpression product or fragment thereof. The expression product can be apart of an intact cell, membrane preparation or lipidic preparation,optionally attached to a support, for example beads, a column, or aplate. The ligand may be contacted with the ion channel peptide before,simultaneously with or after the test compound. Optionally othermolecules essential for the function of the ion channel may be present.Ion channel opening is detectable with the addition of Ion channelsensitive dye, such dyes are known to those in the art. Examples areprovided in Molecular Probes handbook and references therein (MolecularProbes, Inc., 4849 Pitchford Ave, Eugene, USA) and Glaxo Pocket Guide toPharmacology (Dr Michael Sheenal Pharmacology Division Staff, GlaxoGroup Research Ltd., Ware, Herts SG12 0DP). Binding of the test compoundto the ion channel protein modifies its ability to allow ion moleculesacross a membrane. The difference between the observed amount ofmovement of ions across a membrane relative to the theoretical maximumamount of ions that can move across the membrane is a reflection of themodulatory effect of the test compound.

[0125] The invention therefore also relates to a method of screeningcompounds for their ability to alleviate pain, which method comprises:

[0126] (a) contacting at least one test compound in the presence ofvoltage potential sensing dye with a cell containing at least 1 copy ofan expression product of any of the nucleotide sequences of Table 1which is an ion channel protein or with a lipidic matrix containing atleast one copy of the expression product;

[0127] (a) applying a ligand or stimulus;

[0128] (b) determining the opening of the ion channel, and

[0129] (c) selecting a test compound on the basis of its ability tomodulate movement of ions across the membrane.

[0130] e) Receptor Assays

[0131] An expression product of any of the nucleic acid sequences ofTable 1 which encodes a receptor is amenable to screening using receptorassay technology.

[0132] Receptors are membrane associated proteins that initiateintracellular signalling upon ligand binding. Therefore, theidentification of molecules for the prevention and treatment of pain canbe achieved with the use of a ligand-binding assay, as outlined above.Such an assay would utilize an endogenous or non-endogenous ligand as acomponent of the ligand-binding assay. The binding of this ligand to thereceptor in the presence of one or more test compounds would be measuredas described above. Such is the nature of receptors that the assay isusually, but not exclusively performed with a receptor as an intact cellor membranous preparation.

[0133] The invention therefore includes a method of screening for painalleviating compounds, comprising:

[0134] (a) contacting a test compound or test compounds in the presenceof a ligand with cells expressing at least one copy of the expressionproduct of any of the nucleotide sequences of Table 1 which is areceptor or with a lipidic matrix containing at least one copy of theexpression product;

[0135] (b) determining the binding of the test compound to theexpression product, and

[0136] (c) selecting test compounds on the basis of their bindingabilities.

[0137] Transporter Protein Assays

[0138] An expression product of any of the nucleic acid sequences ofTable 1 which encodes a transporter protein which is amenable toscreening using transporter protein assay technology. Non limitingexamples of technologies and methodologies are given by Carroll F I, etal (1995, Medical Research Review, Sep. 15 (5) p 419-444), Veldhuis J Dand Johnson Ml (1994, Neurosci. Biobehav Rep., winter 18(4) 605-12),Hediger M A and Nussberger S (1995, Expt Nephrol, July-August 3(4) p211-218, Endou H and Kanai Y, (1999, Nippon Yakurigaku Zasshi, October114 Suppl 1:1p-16p), Olivier B et al (2000, Prog. Drug Res., 54,59-119), Braun A et al (2000, Eur J Pharm Sci, October 11, Supply 2S51-60) and Molecular Probes handbook and references therein (MolecularProbes, Inc., 4849 Pitchford Ave, Eugene, USA).

[0139] The main function of transporter proteins is to facilitate themovement of molecules across a cellular membrane. Compounds thatmodulate the activity of transporter proteins can be determined with atransporter protein functional screen. Functional screening formodulators of transporter proteins comprises contacting at least onetest compound with an expression product as aforesaid which is atransporter protein and determining the ability of said test compound tomodulate the activity of said transporter protein. The expressionproduct can be part of an intact cell, or lipidic preparation,optionally attached to a support, for example beads, a column or aplate. Binding is preferably performed in the presence of the moleculeto be transported, which should only able to pass through a cellmembrane or lipidic matrix with the aid of the transporter protein. Themolecule to be transported should be able to be followed when it movesinto a cell or through a lipidic matrix. Preferably the molecule to betransported is labelled to aid in characterization, e.g. with achromophore, radioactive, fluorescent, phosphorescent, enzymatic orantibody label. If the molecule to be transported is not directlydetectable it should be amenable to detection and quantification bysecondary detection, which may employ the above technologies. Themolecule to be transported may be contacted with the transporter proteinbefore, simultaneously with or after the test compound. If the bindingof the test compound to the transporter protein modifies its ability totransport molecules through a membranous or lipidic matrix, then thedifference between the observed amount of transported molecule in acell/or through a lipidic matrix relative to the theoretical maximumamount is a reflection of the modulatory effect of the test compound.

[0140] The invention further provides a method for screening compoundsfor their ability to relieve pain, comprising

[0141] a) contacting at least one test compound in the presence oftransporter molecules with a cell containing at least one copy of anexpression product of any of the nucleotide sequences of Table 1 whichis a transporter protein or with a lipidic matrix containing at leastone copy of the expression product;

[0142] c) measuring the movement of transported molecules into or fromthe cell, or across the lipidic matrix; and

[0143] d) selecting test compounds on the basis of their ability tomodulate the movement of transported molecules.

[0144] DNA-Binding Protein Assays

[0145] An expression product of any of the nucleic acid sequences ofTable 1 that encodes a DNA-binding protein is amenable to screeningusing DNA-binding protein assay technology.

[0146] DNA binding proteins are proteins that are able to complex withDNA. The complexing of the DNA binding protein with the DNA in someinstances requires a specific nucleic acid sequence. Screens can bedeveloped in a similar manner to ligand binding screens as previouslyindicated and will utilise DNA as the ligand. DNA-binding protein assayscan be carried using similar principles described in ligand bindingassays as described above. Non limiting examples of methodology andtechnology can be found in the teachings of Haukanes B I and Kvam C(Biotechnology, Jan 11, 1993 60-63), Alberts B et al (Molecular Biologyof the Cell, 1994, 3^(rd) Edn., Garland Publications Inc, Kirigiti P andMachida C A (2000 Methods Mol Biol, 126, 431-51) and Molecular Probeshandbook and references therein (Molecular Probes, Inc., 4849 PitchfordAve., Eugene, USA).

[0147] The invention therefore includes a method of screening for painalleviating compounds, comprising:

[0148] (a) contacting a test compound or test compounds in the presenceof a plurality of nucleic acid sequences with an expression product ofany of the nucleic acid sequences of Table 1 which is a DNA bindingprotein or with cell expressing at least one copy of the expressionproduct or with a lipidic matrix containing at least one copy of theexpression product;

[0149] (b) determining the binding of the test compound to theexpression product, and

[0150] (c) selecting test compounds on the basis of their bindingabilities.

[0151] In the above method, the plurality of nucleic acid sequence maybe added prior to, simultaneously with or after contact of the testcompound with the expression product.

[0152] Oxidoreductases

[0153] Oxidoreductases are enzymes that catalyse the transfer ofhydrogen or oxygen atoms or electrons. These enzymes can by sub-groupedinto twenty categories according to their specific mode of action. Thesegroups are oxidoreductases acting on the CH—OH group of donors (E.C. No1.1), oxidoreductases acting on the aldehyde or oxo group of donors(E.C. No 1.2), oxidoreductases acting on the CH—CH group of the donor(E.C. No 1.3), oxidoreductases acting on the CH-NH2 group of donors(E.C. 1.4), oxidoreductases acting on the CH—NH group of donor (E.C.1.5), oxidoreductases acting on NADH or NADPH (E.C. No 1.6),oxidoreductases acting on other nitrogen compounds as donors (E.C. No1.7), oxidoreductases acting on a sulphur group of donors (E.C. No 1.8),oxidoreductases acting on a haem group of donors (E.C. No 1.9),oxidoreductases acting on diphenols and related substances as donors(E.C. 1.10), oxidoreductases acting on hydrogen peroxide as acceptor(E.C. No 1.11), oxidoreductases acting on hydrogen as donor (E.C. No1.12), oxidoreductases acting on single donors with incorporation ofmolecular oxygen (E.C. No 1.13), oxidoreductases acting on paired donorswith incorporation of molecular oxygen (E.C. No 1.14), oxidoreductasesacting on superoxide radicals as acceptors (E.C. 1.15), oxidoreductasesoxidizing metal ions (E.C. No 1.16), oxidoreductases acting on -CH2groups (E.C. No 1.17), oxidoreductases acting on reduced ferredoxin asdonor (E.C. No 1.18), oxidoreductases acting on reduced flavodoxin asdonor (E.C. No 1.19) and other oxidoreductases (E.C. No 1.97)(Analytical Biochemistry 3^(rd) Edn, David J. Holme and Hazel Peck,Longman Press). The enzyme commission number (E.C.) of the InternationalUnion of Biochemistry relates to the type of reaction catalysed by theenzyme. Further teachings on how to develop assays and screens foroxidoreductases can be obtained from Methods in Enzymology (AcademicPress) with special reference to volume 249.

[0154] Hydrolases

[0155] Hydrolases are enzymes that catalyse hydrolytic reactions and aresub-grouped into eleven classes according to the type of reaction theycarry out. Hydrolases acting on ester bonds (E.C. No 3.1), hydrolasesacting on glycosyl compounds (E.C. No 3.2), hydrolases acting on etherbonds (E.C. No 3.3), hydrolases acting on peptide bonds (E.C. No 3.4),hydrolases acting on carbon-nitrogen bonds, other than peptide bonds(E.C. No 3.5), hydrolases acting on acid anhydrides (E.C. No 3.6),hydrolases acting on acid anhydrides (E.C. No 3.6), hydrolases acting oncarbon-carbon bonds (E.C. No 3.7), hydrolases acting on halide bonds(E.C. No 3.8), hydrolases acting on phosphorous-nitrogen bonds (E.C. No3.9), hydrolases acting on sulphur-nitrogen bonds (E.C. No 3.10) andhydrolases acting on carbon-phosphorous bonds (Analytical Biochemistry3^(rd) Edn, David J. Holme and Hazel Peck, Longman Press). The enzymecommission number (E.C.) of the International Union of Biochemistryrelates to the type of reaction catalysed by the enzyme. Furtherteachings on how to develop assays and screens for hydrolases can beobtained from Methods in Enzymology (Academic Press) with specialreference to volume 249.

[0156] C) In vivo Functional Screen

[0157] Any of the nucleotide sequences of Table 1 or homologues thereofmay be inserted by means of an appropriate vector into the genome of alower vertebrate or of an invertebrate animal or may be inactivated ordown regulated in the genome of said animal. The resulting geneticallymodified animal may be used for screening compounds for effectiveness inthe regulation of pain. The invertebrate may, for example, be a nematodee.g. Caenorhabditis elegans, which is a favourable organism for thestudy of response to noxious stimuli. Its genome sequence has beendetermined, see Science, 282, 2012 (1998), it can be bred and handledwith the speed of a micro-organism (it is a self-fertilizinghermaphrodite) and can therefore be used in a high throughput screeningformat (WO 00/63424, WO 00/63425, WO 00/63426 and WO 00/63427), and itoffers a full set of organ systems, including a simple nervous systemand contains many similarly functioning genes and signaling pathways tomammals. A thermal avoidance model based on a reflexive withdrawalreaction to an acute heat stimulus has been described by Wittenburg etal, Proc. Natl. Acad. Sci. USA, 96, 10477-10482 (1999), and allows thescreening of compounds for the treatment of pain with the modulation ofpain sensation as an endpoint.

[0158] The genome of C. elegans can be manipulated using homologousrecombination technology which allows direct replacement of nucleicacids encoding C. elegans with their identified mammalian counterpart.Replacement of these nucleic acids with those nucleic acids outlinedabove would allow for the direct screening of test compound(s) withtheir expression products. Any of the pain-related genes described abovemay be ligated into a plasmid and introduced into oocytes of the worm bymicroinjection to produce germline transformants. Successful plasmidinjection into C. elegans and expression of inserted sequences has beenreported by Devgen B. V., Ghent, Belgium. It is also possible to produceby routine methods worms in which the target sequences aredown-regulated or not expressed (knock-out worms). Further non limitingexamples of methodology and technology can be found in the teachings ofHazendonk et al (1997, Nat genet. 17(1) 119-21), Alberts et al, (1994,Molecular Biology of the Cell 3^(rd) Ed. Garland Publishing Inc,Caenorhabditis elegans is anatomically and genetically simple),Broverman Set al, (1993, PNAS USA 15; 90(10) 4359-63) and Mello et al(1991, 10(12) 3959-70).

[0159] A further method for screening compounds for ability to modifyresponse to pain, e.g. relieve pain, comprises:

[0160] (a) contacting one or more test compounds with at least one Celegans containing at least one copy of a sequence as set out above;

[0161] (b) subjecting the C. elegans to a nociceptive stimulus;

[0162] (c) observing the response of the C. elegans to said stimulus;and

[0163] (d) selecting test compounds on the basis of their ability tomodify the response of C. elegans to said stimulus.

[0164] Diagnostic Tools and Kits

[0165] Affinity Peptides, Ligands and Substrates

[0166] Pain associated polypeptides and fragments thereof can bedetected at the tissue and cellular levels with the use of affinitypeptides, ligands and substrates, which will enable a skilled person todefine more precisely a patient's ailment and help in the prescriptionof a medicament. Such affinity peptides are characterized in thatfirstly they are able to bind specifically to a pain associatedpolypeptide, and secondly that they are capable of being detected. Suchpeptides can take the form of a peptide or polypeptide for example anantibody domain or fragment, or a peptide/polypeptide ligand orsubstrate, or a polypeptide complex such as an antibody.

[0167] The preparation of such peptides and polypeptides are known tothose in the art. Antibodies, these may be polyclonal or monoclonal, andinclude antibodies derived from immunized animals or from hybridomas, orderivatives thereof such as humanized antibodies, Fab or F(ab′)2antibody fragments or any other antibody fragment retaining the antigenbinding specificity.

[0168] Antibodies directed against pain-associated gene productmolecules may be produced according to conventional techniques,including the immunization of a suitable mammal with the peptides orpolypeptides or fragment thereof. Polyclonal antibodies can be obtaineddirectly from the serum of immunized animals. Monoclonal antibodies areusually produced from hybridomas, resulting from a fusion betweensplenocytes of immunized animals and an immortalized cell line (such asa myeloma). Fragments of said antibodies can be produced by proteasecleavage, according to known techniques. Single chain antibodies can beprepared according to the techniques described in U.S. Pat. No.4,946,778. Detection of these affinity peptides could be achieved bylabeling. Technologies, which allow detection of peptides, such asenzymatic labeling, fluorescence labeling or radiolabeling are wellknown to those in the art. Optionally these affinity peptides, ligandsand substrates could themselves be detected with the use of a moleculethat has specific affinity to the peptides, ligands and substrates andis itself labeled.

[0169] The invention further provides a kit comprising;

[0170] (a) affinity peptide and/or ligand and/or substrate for anexpression product of a gene sequence that is up-regulated in the spinalcord of a mammal in response to first and second models of neuropathicor central sensitization pain; and

[0171] (b) a defined quantity of an expression product of a genesequence that is up-regulated in the spinal cord of a mammal both inresponse to first and second models of neuropathic or centralsensitization pain,

[0172] for simultaneous, separate or sequential use in detecting and/orquantifying an expression product of a gene sequence that isup-regulated in the spinal cord of a mammal in response to first andsecond models of neuropathic or central sensitization pain.

[0173] Complimentary nucleic acids

[0174] Pain associated nucleic acid sequences can be characterized atthe tissue and cellular levels with the use of complimentary nucleicacid sequences. Detection of the level of expression of pain associatednucleic acid sequences can help in the prognosis of a pain condition andthe prescription of a medicament. These complimentary nucleic acids arecharacterized in that they can hybridize to a pain associated nucleicacid sequence and their presence can be detected through varioustechniques. Such techniques are known to those in the art and mayinclude detection by polymerase chain reaction or detection by labelingof complimentary nucleic acid sequences by enzymatic labeling, affinitylabeling fluorescent labeling or radiolabeling. Complimentary strandnucleic acid sequences of this invention are 10 to 50 bases long, morepreferably 15 to 50 bases long and most preferably 15 to 30 bases long,and hybridize to the coding sequence of the nucleic acid sequences.

[0175] A further aspect of this invention is a kit that comprises:

[0176] (a) nucleic acid sequences capable of hybridization to a nucleicacid sequence that is up-regulated in the spinal cord of a mammal inresponse to first and second models of neuropathic or centralsensitization pain; and

[0177] (b) a defined quantity of one or more nucleic acid sequencescapable of hybridization to a nucleic acid sequence that is up-regulatedin the spinal cord of a mammal in response to first and second models ofneuropathic or central sensitization pain, for simultaneous, separate orsequential use in detecting and/or quantifying a gene sequence that isup-regulated in the spinal cord of a mammal in response to first andsecond models of neuropathic or central sensitization pain.

[0178] Identification and Validation

[0179] Subtractive hybridization enables the identification of nucleicacid sequences whose expression profiles are modified by a stimulus.Upon stimulation of a system (in the case of this invention anociceptive stimulus on an animal model) all observed changes in thelevel of nucleic acid sequence expression are due to the reaction of thesystem to the stimulus. Characterization of these changes in expressionby way of identification of nucleic acid sequence and level ofexpression is both identification and validation.

[0180] The inventors have developed a four step process which allows forthe simultaneous identification and validation of nucleic acid sequenceswhose expression are regulated by a pain stimulus, preferably a chronicpain stimulus, and more preferably a diabetic pain stimulus. Thisprocess may comprise the following steps:

[0181] (a) induction of a nociceptive stimulus in test animals;

[0182] (b) extraction of nucleic acids from specific neuronal tissue oftest and control animals;

[0183] (c) selective amplification of differentially expressed nucleicacid sequence; and

[0184] (d) identification and characterization of differentiallyexpressed gene products that are modulated by a nociceptive stimulus.

[0185] The above process is described in more detail below.

[0186] (a) Induction of Nociceptive Stimulus

[0187] The effect of the selected nociceptive stimulus on the testanimal needs to be confirmable. The test subjects are therefore aspecies that has a “developed” nervous system, preferably similar tothat of humans, most preferably rats or mice. Advantageously, thenociceptive stimulus is analogous to known pain paradigms in humans. Onesuch paradigm of pain is the pain associated with diabetes, which can beinduced in rodents with the use of streptozotocin (STZ). The presentapplication requires the sequences to be up-regulated in two painmodels, and mechanical damage can provide an appropriate second model.

[0188] Streptozotocin (STZ) induces hyperglycemia and Type 1 diabetesmellitus in rats. In particular, STZ contains a glucose analogue thatallows it to be taken up by the glucose transporter 2 present on thesurface of pancreatic β cells, the site of insulin synthesis. Onceinside the cell, STZ causes a reduction in the level of nicotinamideadenine dinucleotide (NAD⁺). The decrease in NAD⁺ levels eventuallyleads to necrosis of the pancreatic β cell, causing a reduction ininsulin levels and then diabetes, leading to neuropathy (diabetic) andneuropathic pain (R. B. Weiss, Cancer Treat. Rep., 66, 427-438 1982, Guyet al, Diabetologica, 28, 131-137 1985; Ziegler et al, Pain, 34, 1-101988; Archer et al, J. Neural. Neurosurgeon. Psychiatry, 46, 491-4991983). The diabetic rat model has been shown to be a reliable model ofhyperalgesia. We have used an STZ-induced diabetic rat model to create astate of hyperlagesia that can be compared with control animals(Courteix et al, Pain, 53, 81-88 1993).

[0189] Three models of neuropathic pain in rats, which involve nerveinjury, may be used, see Ralston, D D (1998) Present models ofneuropathic pain. Pain Rev. 5: 83-100. The injuries are caused by (1)loosely tying four chromic gut sutures around the sciatic nerve (CCImodel developed by Bennett, G J and Y-K Xie, A peripheral mononeuropathyin rat that produces disorders of pain sensation like those seen in man,Pain 33: 87-107, 1988), (2) tightly ligating one third to one half ofthe fibers in the sciatic nerve (model developed by Seltzer, Z, RDubner, Y Shire, A novel behavioral model of neuropathic pain disordersproduced in rats by partial sciatic nerve injury, Pain 43: 205-218,1990), and (3) tightly ligating the dorsal spinal nerve of a rat at theL5 or L5 and L6 levels (L5 model developed by Kim, S H and J M Chung, Anexperimental model for peripheral neuropathy produced by segmentalspinal nerve ligation in the rat, Pain 50:355-363, 1992).

[0190] (b) Extraction of Nucleic Acids from Neuronal Tissue of Test andControl Animals

[0191] The inventors have determined that RNA extraction of whole spinalcord nervous tissue would provide a way of identifying nucleic acidsequences whose expression in spinal tissue is modulated bystreptozotocin induced diabetes or by a mechanical nerve damage modelfor neuropathic pain e.g. CCI. Test (subjected to the nociceptivestimulus) and control animals were sacrificed, and the tissue to bestudied e.g. neural tissue separated. Techniques for so doing varywidely from animal to animal and will be familiar to skilled persons.

[0192] A cDNA library can be prepared from total RNA extracted fromneural tissue of the test and control animals. Where possible, however,it is preferred to isolate the mRNA from the total RNA of the test andcontrol animals, by affinity chromatography on oligo (dT)-cellulose, andthen reverse transcribe the mRNA from the test and control animals togive test and control cDNA. Converting mRNA from the test and controlanimals to corresponding cDNA may be carried out by any suitable reversetranscription method, e.g. a method as described by Gubler & Hoffman,Gene, 25, 263-269 (1983). If desired a proprietary kit may be used e.g.the CapFinder PCR cDNA Library Construction Kit (Life Technologies)which is based on long-distance PCR and permits the construction of cDNAlibraries from nanograms of total RNA.

[0193] (c) Selective Amplification of Differentially Expressed NucleicAcids

[0194] The reverse transcribed cDNA of the test and control animals issubjected to subtractive hybridisation and amplification so thatdifferentially expressed sequences become selectively amplified andcommonly expressed sequences become suppressed, so as to over-produceDNA associated with said nociceptic stimulus. A wide range ofsubtractive hybridisation methods can be used, but the preferred methodis so-called suppression subtractive hybridisation, see U.S. Pat. No.5,565,340 and Diatchenko et al, Proc. Nat. Acad. Sci. USA, 93, 6025-6030(1996), the disclosures of which are herein incorporated by reference.Kits for carrying out this method are available from CLONTECHLaboratories, Inc.

[0195] (d) Cloning and Sequencing the Differentially Expressed cDNA

[0196] The differentially expressed cDNA is ligated into a cloningvector, after which cells of E. coli are transformed with the vector andcultured. Positive clones are selected and lysed to release plasmidscontaining the cDNA insert. The plasmids are primed using forward andreverse primers to either side of the cloning site and the cDNA insertis sequenced. Vector and adaptor sequences are then removed from theoutput data from the sequencer, leaving only the nucleotide sequence ofthe differentially expressed gene. The sequence is then checked againstdata held in a database for homology to known nucleotide sequencesincluding expressed sequence tags (ESTs) and coding sequences forproteins.

[0197] (e) Validation of the Above Method

[0198] The importance of the sequences that we have identified in painis confirmed by the fact that genes that represent nucleic acidsequences which have previously been implicated in pain, includingCalmodulin (pRCM1, Genebank X13933), Enkephalin (Genebank Y07503) andNeurotensin receptor type 2 (Genebank X97121) have also been identifiedusing this method.

[0199] The inventors have identified nucleic acid sequences of the MAPkinase pathway, a previously non pain-associated biological pathway. Theinventors have subsequently shown that intra-spinal injection of a MEKinhibitor (MEK is part of the MAP kinase pathway) produces a powerfulinhibition of pain (Patent application No U.S. 60/144,292).Subsequently, it has been shown that the MAP kinase is also implicatedin acute inflammatory pain (Woolf et al, Nature Neuroscience 1999).

[0200] The invention will now be further described in the followingExample.

EXAMPLE

[0201] Induction of Diabetes

[0202] Diabetes was induced in adult (150-200 g) male Sprague-Dawleyrats (n=6) as described by Courteix et al (supra). Animals were injectedintraperitoneally with streptozotocin (STZ)(50 mg/kg) dissolved indistilled water. Control or sham animals (age-matched animals, n=6) wereinjected with distilled water only.

[0203] Chronic Constrictive Injury (CCI)

[0204] Rats were anaesthetized with i.p. sodium phenobarbital, afterwhich the common left sciatic nerve was exposed at the level of themiddle of the thigh by blunt dissection through the biceps femoris andproximal to the sciatic trifurcation. Four ligatures (4.0 braided silk)were tied loosely around it with about 1 mm spacing. The muscle wasclosed in layers and two wound clips were applied to close the skinincision. The wound was then covered with topical antibiotics.

[0205] Nociceptive Testing

[0206] Static allodynia (a form of hyperlagesia) was measured using amethod described by Chaplan let al, “Quantitative assessment of tactileallodinya in the rat paw”, J. Neurosci. Methods, 53, 55-63 (1994). Aseries of von Frey filaments of different buckling weight (i.e. the loadrequired for the filament to bend) were applied to the plantar surfaceof the right hand paw. The starting filament had a buckling weight of 20g. Lifting of the paw was taken to be a positive result, in which case afilament with the next lowest buckling weight was used for the nextmeasurement. The test was continued until a filament was found for whichthere was an absence of response for longer than 5 seconds whereas are-test with the next heaviest filament gave a positive response.Animals were considered hyperalgesic if their thresholds were found tobe <4 g of those of comparable untreated rats, see Calcutt & Chaplan,“Spinal pharmacology of tactile allodynia in diabetic rats”, British J.Pharmacol, 122, 1478-1482 (1997).

[0207] Tissue Extraction

[0208] STZ-treated, CCI-treated and control animals were anaesthetizedwith 4% halothane and perfused with ice-cold 0.9% saline containing 1%citric acid (pH adjusted to 7.4 with NaOH). The animals were decapitatedand the lumbar spinal cord exposed. A 2-centimetre length of spinal cordending at L6 (lumbar-6 forward) was removed from the spinal column.Attached dorsal root ganglia and contaminating spinal connective tissueswere removed. The spinal cord tissue was snap frozen in dry ice andisopentane. In the experiments that follow, procedures on thestreptozocin-treated and control groups of animals are disclosed. Itwill be understood that for identification of CCI-treated animals thesame experiments are performed, but using tissues from the CCI-treatedanimals and from control animals.

[0209] Total RNA Extraction

[0210] Total RNA was extracted from the pooled male rat tissues of thestreptozocin-treated and control groups using the TRIZOL Reagent Kit(Life Technologies). Briefly, tissue samples were homogenised fullyusing a Polytron homogenizer in 1 ml of TRIZOL reagent per 50-100 mg oftissue. Homogenized samples were then incubated at room temperature for5 minutes and phase separated using 0.2 ml chloroform per 1 ml TRIZOLreagent followed by centrifugation at 3,000 g. The aqueous phase wastransferred to a fresh tube and the RNA precipitated with an equalvolume of isopropyl alcohol and followed by centrifugation at 10,000 g.The RNA pellet was washed in 75% ethanol and re-centrifuged. The pelletwas then air dried and re-suspended in water.

[0211] mRNA Extraction

[0212] In contrast to ribosomal RNA and transfer RNA, the vast majorityof mRNAs of mammalian cells carry tracts of poly(A+) at their 3′termini. mRNAs can therefore be separated from the bulk of cellular RNAby affinity chromatography on oligo (dT)-cellulose. mRNA was extractedfrom Total RNA using the MESSAGEMAKER Kit (Life Technologies) in whichmRNA (previously heated to 65° C. in order to disrupt secondarystructures and so expose the poly (A⁺) tails) was bound to oligo (dT)cellulose under high salt concentrations (0.5M NaCl) in a filtersyringe. Unbound RNA was then washed away and the poly(A⁺) mRNA elutedin distilled water. A tenth of the volume of 7.5 M Ammonium Acetate, 50μg of glycogen/ml mRNA sample and 2 volumes of absolute alcohol werethen added to the samples which are then placed at −20° C. overnight.Following precipitation, the mRNA was spun down at 12,000 g for 30minutes at 4° C. RNAase free water was used to re-suspend the pellets,which were then and stored at −80° C.

[0213] PCR Select

[0214] The technique used was based on that of the CLONTECH PCR SelectSubtraction Kit. The following protocol was performed using STZ-treatedlumbar spinal cord Poly A⁺ RNA as the ‘Tester’ and Sham lumbar spinalcord poly A⁺ RNA as the ‘Driver’ (Forward Subtraction). A secondsubtraction experiment using the Sham lumbar spinal cord mRNA as the‘Tester’ and STZ treated lumbar spinal cord mRNA as the ‘Driver’(Reverse Subtraction) was performed in parallel using the same reagentsand protocol. As a control for both experiments, the subtraction wasalso carried out using human skeletal muscle mRNA that had been providedby the kit manufacturer.

[0215] First-Strand cDNA Synthesis

[0216] 2 μg of PolyA⁺ RNA and 1 μl of cDNA synthesis primer (10 μM) werecombined in a 0.5 ml Eppendorf tube and sterile H₂O was added wherenecessary to achieve a final volume of 5 μl. The contents were mixedgently and incubated in a thermal cycler at 70° C. for 2 min. The tubeswere then cooled on ice for two minutes, after which 2 μl of5×First-strand buffer, 1 μl of dNTP mix (10 mM each), sterile H₂O and 1μl of AMV reverse transcriptase (20 units/μl) was also added. The tubeswere then placed at 42° C. for 1.5 hr in an air incubator. First-strandcDNA synthesis was terminated by placing the tubes on ice. (the humanskeletal muscle cDNA produced by this step was used as the ‘controldriver’ in later steps).

[0217] Second-Strand cDNA Synthesis

[0218] 48.4 μl of Sterile H₂O, 16.0 μl of 5×Second-strand buffer, 1.6 μlof dNTP mix (10 mM) and 4.0 μl of 20×second-strand enzyme cocktail wereadded to each of the first-strand synthesis reaction tubes. The contentswere then mixed and incubated at 16° C. in a thermal cycler for 2 hr. 6units (2 μl) of T4 DNA polymerase was then introduced and the tubes wereincubated for a further 30 min at 16° C. In order to terminatesecond-strand synthesis, 4 μl of 20×EDTA/glycogen mix was added. Aphenol:chloroform extraction was then carried out using the followingprotocol:

[0219] 100 μl of phenol:chloroform:isoamyl alcohol (25:24:1) was addedto the tubes which were then vortexed thoroughly and centrifuged at14,000 rpm for 10 min at room temperature. The top aqueous layer wasremoved and placed in a fresh tube. 100 ill of chloroform:isoamylalcohol (24:1) was then added to the aqueous layer and the tubes wereagain vortexed and centrifuged at 14,000 rpm for 10 min. 40 μl of 4 MNH₄OAc and 300 μl of 95% ethanol were then added and the tubescentrifuged at 14,000 rpm for 20 min. The supernatant was removedcarefully, then 500 μl of 80% ethanol was added to the pellet. The tubeswere centrifuged at 14,000 rpm for 10 min and the supernatant wasremoved so that the pellet could be air-dried. The precipitate was thendissolved in 50 μl of H₂O. 6 μl was transferred to a freshmicrocentrifuge tube. The remainder of the sample was stored at −20° C.until needed.

[0220] Rsa I Digestion

[0221] All products of the above procedures were subjected to arestriction digest, using the restriction endonuclease Rsa I, in orderto generate ds cDNA fragments that are short and thus are optimal forsubtraction hybridisation due to the standard kinetics of thehybridisation. Also, as Rsa I makes a double stranded cut in the middleof a recognition sequence, ‘blunt ends’ of a known nucleotide sequenceare produced allowing ligation of adaptors onto these ends in a laterstep. The following reagents were added to the 6 μl product of thesecond hybridisation (see above): 43.5 μl of ds cDNA, 5.0 μl 10×Rsa Irestriction buffer and 1.5 μl of Rsa I (10 units/μl). The reactionmixture was incubated at 37° C. for 1.5 hr. 2.5 μl of 20×EDTA/glycogenmix was used to terminate the reaction. A phenol:chloroform extractionwas then performed as above (second-strand cDNA synthesis section). Thepellet produced was then dissolved in 5.5 μl of H₂O and stored at −20°C. until needed. The preparation of the experimental ‘Driver’ cDNAs andthe control skeletal muscle cDNA was thus completed.

[0222] Adaptor Ligation

[0223] The adaptors were not ligated to the driver cDNA.

[0224] 1 μl of each Rsa I-digested experimental cDNA (from the Rsa IDigestion above) was diluted with 5 μl of sterile water. Preparation ofthe control skeletal muscle tester cDNA was then undertaken by brieflycentrifuging the tube containing control DNA (Hae III-digest of φX174DNA [3 ng/μl]) and diluting 2 μl of the DNA with 38 μl of sterile water(to 150 ng/ml). 1 μl of control skeletal muscle cDNA (from the Rsa IDigestion) was then mixed with 5 μl of the diluted φX174/Hae III DNA(150 ng/ml) in order to produce the control skeletal muscle tester cDNA.

[0225] Preparation of the Adaptor-Ligated Tester cDNA

[0226] A ligation master mix was prepared by combining 3 μl of sterilewater, 2 μl of 5×ligation buffer and 1 μl T4 DNA ligase (400 units/μl)per reaction. 2 μl of adaptor 1 (10 μM) was then added to 2 μl of thediluted tester cDNA. To this, 6 μl of the ligation master mix was alsoadded. The tube was therefore labeled Tester 1-1. In a separate tube, 2μl of the adaptor 2R (10 μM) was mixed with 2 μl of the diluted testercDNA and 6 μl of the master mix. This tube was named Tester 1-2.

[0227] 2 μl of Tester 1-1 and 2 μl of Tester 1-2 were then placed intofresh tubes. These would later be used as the unsubtracted testercontrol. The remainder of the contents of Tester 1-1 and Tester 1-2tubes were then centrifuged briefly and incubated at 16° C. overnight.The ligation reaction was stopped by adding 1 μl of EDTA/glycogen mixand the samples were heated at 72° C. for 5 min in order to inactivatethe ligase. In doing so, preparation of the experimental and controlskeletal muscle adaptor-ligated tester cDNAs was complete.

[0228] 1 μl from each unsubtracted tester control was then removed anddiluted into 1 ml of water. These samples were set aside as they were tobe used later for PCR (see below). All of the samples were stored at−20° C.

[0229] Analysis of Ligation Efficiency

[0230] 1 μl of each ligated cDNA was diluted into 200 μl of water andthe following reagents were then combined in four separate tubes: Tube:Component 1 2 3 4 Tester 1-1 (ligated to Adaptor 1) 1 1 — — Tester 1-2(ligated to Adaptor 2R) — — 1 1 G3PDH 3′ primer(10 μM) 1 1 1 1 G3PDH 5′primer(10 μM) — 1 — 1 PCR primer 1 (10 μM) 1 — 1 — Total volume μl 3 3 33

[0231] A master mix for all of the reaction tubes plus one additionaltube was made up by adding 18.5 μl of sterile H₂O, 2.5 μl of 10×PCRreaction buffer, 0.5 μl of dNTP mix (10 mM), and 0.5 μl of 50×AdvantagecDNA Polymerase Mix, per reaction, into a fresh tube. 22 μl of thismaster mix was then aliquotted into each of the 4 reaction tubesprepared above. The contents of the tubes were overlaid with 50 μl ofmineral oil. The reaction mix was incubated in a thermal cycler at 75°C. for 5 min in order to extend the adaptors. The following protocol wasthen carried out immediately in a thermal cycler (Perkin-Elmer GeneAmpPCR Systems 2400): 94° C. for 30 sec (1 cycle), 94° C. 10 sec, 65° C. 30sec and then 68° C. 2.5 min (25 cycles)

[0232] First Hybridisation

[0233] 1.5 μl of the Adaptor 1-ligated Tester 1-1 was combined with 1.5μl of the Rsa I-digested driver cDNA, prepared earlier and 1 μl of4×Hybridisation buffer. This process was then repeated combining theAdaptor 2R-ligated Tester 1-2 with the Rsa I-digested driver cDNA and4×hybridisation buffer. The samples were incubated in a thermal cyclerat 98° C. for 1.5 min followed by incubation at 68° C. for 8 hr.

[0234] Second Hybridisation

[0235] 1 μl of Driver cDNA (i.e. the Rsa I-digested cDNA (see above)), 1μl 4×Hybridisation buffer and 2 μl Sterile H₂O were all combined in afresh tube. 1 μl of this mix was then removed and placed in a new tube,overlaid with 1 drop of mineral oil and incubated at 98° C. for 1.5 minin order to denature the driver. The following procedure was used tosimultaneously mix the driver with hybridisation samples 1 and 2(prepared in the first hybridisation), thus ensuring that the twohybridisation samples were mixed together only in the presence offreshly denatured driver: A micropipettor was set at 15 pl. The pipettetip was then touched onto the mineral oil/sample interface of the tubecontaining hybridisation sample 2. The entire sample was drawn partwayinto the tip before it was removed from the tube in order to draw asmall amount of air into the tip. The pipette tip was then touched ontothe interface of the tube containing the freshly denatured driver (i.e.the tip contained both samples separated by a small pocket of air)before the entire mixture was transferred to the tube containinghybridisation sample 1. The reaction was then incubated at 68° C.overnight. 200 μl of dilution buffer was added to the tube, which wasthen heated in a thermal cycler at 68° C. for 7 min. The product of thissecond hybridisation was stored at −20° C.

[0236] PCR Amplification

[0237] Seven PCR reactions were set up: (1) The forward-subtractedexperimental cDNA, (2) the unsubtracted tester control (see preparationof the adaptor ligated tester cDNA), (3) the reverse-subtractedexperimental cDNA, (4) the unsubtracted tester control for the reversesubtraction, (5) the subtracted control skeletal muscle cDNA, (6) theunsubtracted tester control for the control subtraction, and (7) the PCRcontrol subtracted cDNA (provided in the kit). The PCR controlsubtracted cDNA was required to provide a positive PCR control as itcontained a successfully subtracted mixture of Hae III-digested φX174DNA.

[0238] The PCR templates were prepared by aliquotting 1 μl of eachdiluted cDNA (i.e., each subtracted sample from the second hybridisationand the corresponding diluted unsubtracted tester control produced bythe adaptor ligation, see above) into an appropriately labeled tube. 1μl of the PCR control subtracted cDNA was placed into a fresh tube. Amaster mix for all of the primary PCR tubes, plus one additionalreaction, was then prepared by combining 19.5 μl of sterile water, 2.5μl of 10×PCR reaction buffer, 0.5 μl of dNTP Mix (10 mM), 1.0 μl of PCRprimer 1 (10 μM) and 0.5 μl of 50×Advantage cDNA Polymerase Mix. 24 μlof Master Mix was then aliquotted into each of the 7 reaction tubesprepared above and the mixture was overlaid with 50 μl of mineral oil,before being incubated in a thermal cycler at 75° C. for 5 min in orderto extend the adaptors. Thermal cycling was then immediately startedusing the following protocol: 94° C. 25 sec (1 cycle), 94° C. 10 sec,66° C. 30 sec and 72° C. 1.5 min (32 cycles).

[0239] 3 μl of each primary PCR mixture was then diluted in 27 μl ofH₂O, 1 μl of each of these dilutions was then placed into a fresh tube.

[0240] A master mix for the secondary PCRs, (plus an additionalreaction) was set up by combining 18.5 μl of sterile water, 2.5 μl of10×PCR reaction buffer, 1.0 μl of Nested PCR primer 1 (10 μM), 1.0 μl ofNested PCR primer 2R (10 μM), 0.5 μl of dNTP Mix (10 mM) and 0.5 μl of50×Advantage cDNA Polymerase Mix per reaction. 24 μl of this Master Mixwas then added into each reaction tube containing the 1 μl dilutedprimary PCR mixture. The following PCR protocol was then carried out:94° C. 10 sec, 68° C. 30 sec and 72° C. 1.5 min (12 cycles). Thereaction products were then stored at −20° C.

[0241] Ligation into a Vector/Transformation & PCR

[0242] The products of the PCR amplification (enriched fordifferentially expressed cDNAs) were ligated into the pCR2.1-TOPO vectorusing a T/A cloning kit (Invitrogen), transformed into TOPO One Shotcompetent cells according to the manufacturers protocol and grown up onLB (Luria-Bertani) Agar plates overnight at 37° C. 1,000 colonies werethen individually picked (using fresh sterile tips) and dipped into 5 μlof sterile water which had been aliquotted previously into 96 well PCRplates. The water/colonies were heated in a thermal cycler at 100° C.for 10 minutes in order to burst the cells, thus releasing the plasmidscontaining a differentially expressed cDNA insert. The 5 μl ofwater/plasmid was then used as a template in a PCR reaction (see below)using M13 Forward and Reverse primers (10 ng/μl), complementary to theM13 site present on either side of the cloning site on the vector. 5 μlof the PCR product was then run on a 2% agarose gel and stained byethidium bromide. PCR products of an amplified insert were identifiedand 5 μl of the remainder of the PCR product (i.e. from the 15 μl thathad not been run on the gel) was diluted {fraction (1/10)} with water. 5μl of the diluted PCR product was then used as a template in asequencing reaction.

[0243] Sequencing

[0244] A sequencing reaction containing M13 primer (3.2 pmol/μl),‘BigDye’ reaction mix (i.e. AmpliTaq® DNA polymerase, MgCl₂, buffer andfluorescent dNTPs [each of the four deoxynucleoside triphosphates islinked to a specific fluorescent donor dye which in turn is attached toa specific acceptor dye]) and cDNA template (diluted PCR product) wasset up. The reaction was carried out on a thermal cycler for 25 cyclesof 10 seconds at 96° C., 20 seconds at 50° C. and 4 minutes at 60° C.Each reaction product was then purified through a hydrated Centri-Sepcolumn, and lyophilised. The pellets were resuspended in TemplateSupression Reagent and sequenced on an ABI Prism 310 Genetic Analyser.The analyser uses an ion laser to excite the specific donor dye thattransfers its energy to the acceptor dye, which emits a specific energyspectrum that can be read by the sequencer.

[0245] The potentially upregulated genes of the streptozocin-induceddiabetes experiment and of the CCI experiment were sequenced atParke-Davis, Cambridge and at the applicant's core sequencing facilityin Ann Arbor, Mich., USA.

[0246] Bioinformatics

[0247] The sequencing results were analysed using the computer programCHROMAS in which the vector and adaptor sequences were clipped off,leaving only the nucleotide sequence of the differentially expressedgene. Each sequence was then checked for homology to known genes,Expressed Sequence Tags (ESTs) and Proteins using various Basic LocalAlignment Search Tool (BLAST) searches against the Genbank sequencedatabase at the National Centre for Biotechnology Information, Bethesda,Md., USA (NCBI).

[0248] Lists were derived called STZup and STZdown and CCIup and CCIdownthat contain the nucleic acid sequences from the forward and backsubtracted libraries respectively. In each list there are givenaccession numbers and descriptions for the known rat genes identified,and where available corresponding mouse or human genes. Sequences thatare considered to be of interest and that are up-regulated both in astreptozocin-induced diabetes model and in a chronic constrictive injurypain model are listed in Table 1 below.

[0249] References have been given where available for the sequences thathave been found, and sequence listings have been given in the form inwhich they currently appear in publicly searchable databases e.g. theNCBI databaase (National Center for Biotechnology Information, NationalLibrary of Medicine, National Institutes of Health, Bethesda, Md.,20894, USA, www.ncbi.nlm.nih.gov). These sequence listings are given forthe purposes of identification only. The invention includes the use ofsubsequently revised versions of the above sequences (which mayincorporate small differences to the version set out herein) andhomologous sequences or similar proteins in other species as determinedby a high percentage identity (e.g. above 50%, preferably above 90%),length of alignment and functional equivalence. Rat Mouse HumanLiterature Expression Accession accession Accession Reference product orname Number number Number Reaction Assay No. Seq. ID No's A-rafoncogene, X06942 U01337 Mitogenic Ser/Thr kinase 1 1 Protein liverexpressed** signaling 2. cDNA Protein D90164 X80910 Cell signalingSer/Thr 2 3. Protein phosphatase 1 phosphatase 4. cDNA PhosphofructokinU25651 AF249894 Y00698 Glycolysis Transferase 3 5. Protein ase, muscle6. cDNA (PFK-M) Alkaline phospho- D28560 AF123542 D45421 MyelinationPhospho- 4 7. Protein diesterase 1* diesterase 8. cDNA Na + K + ATPaseM14512 Membrane Na+/K+ 5 9. Protein alpha + isoform potential transport10. cDNA catalytic subunit Putative vacuolar U13837 AF113129 Ion channelH+ transport 6 11. Protein ATP synthase 12. cDNA subunit AHypoxia-inducible AF057308 AF003695 U22431 DNA binding N/A 7 13. Proteinfactor-1 alpha 14. cDNA (Hifla) Cytochrome-c M64496 EnergyOxidoreductase 8 15. Protein oxidse II, metabolism 16. cDNAmitochondrial Round spermatid U97667 Glutathione Hydrolase 9 17. Proteinprotein RSP29 metabolism Putative 26S Q16401 Proteolysis Traficking of10 18. Protein proteasome transmembrane subunit S5B proteins Nap 1AB014509 Binds to Nck 11 19. Protein in signal 20. cDNA transduction;Nck is involved in cytoskeleton guidance Novel rat cofilin L29468AF134803 Cytoskeleton 12 21. Protein protein 22. cDNA GangliosideAB003575 O08765 Similar to N/A 23. Protein expression factor- BAA human(Human) 2 (GEF-2) 19975 GABA(A) 24. cDNA receptor- (mouse) associatedprotein-like 2 and to mouse protein Putative KIAA Voltage- N/A 25.(Protein) 1117 gated 26. cDNA potassium- channel protein Putative F1-20M83985 Synapse- 13 27. (Protein) phosphoprotein specific 28. cDNAprotein Proliferating cell M24604 DNA repair 14 N/A (withdrawn) nuclearantigen and (PCNA/cyclin) replication Ribosomal protein J02650 Protein15 29. (Protein) L19 synthesis 30. cDNA 14-3-3 protein U37252 D83037Cell N/A 31. (Protein) zeta subtype signalling 32. cDNA 14-3-3 proteineta D17445 U57311 X80536 Signal 16 33. (Protein) subtype transduction.34. cDNA

REFERENCES IN THE TABLE 1

[0250] 1. Ishikawa, F., Takaku, F., Nagao, M. and Sugimura, T., Thecomplete primary structure of the rat A-raf cDNA coding region:conservation of the putative regulatory regions present in rat c-raf,Oncogene Res., 1 (3), 243-253 (1987).

[0251] 2. Sasaki, K., Shima, H., Kitagawa, Y., Irino, S., Sugimura, T.and Nagao, M., Identification of members of the protein phosphatase Igene family in the rat and enhanced expression of protein phosphatase 1alpha gene in rat hepatocellular carcinomas, Jpn. J. Cancer Res. 81(12), 1272-1280 (1990), Erratum: Jpn J Cancer Res July 1991; 82(7), 873.

[0252] 3. Ma, Z., Ramanadham, S., Kempe, K., Hu, Z., Ladenson, J. andTurk, J., Characterization of expression of phosphofructokinase isoformsin isolated rat pancreatic islets and purified beta cells and cloningand expression of the rat phosphofructokinase-A isoform, Biochim.Biophys. Acta, 1308 (2), 151-163 (1996).

[0253] 4. Narita, M., Goji, J., Nakamura, H. and Sano, K., Molecularcloning, expression, and localization of a brain-specificphosphodiesterase I/nucleotide pyrophosphatase (PD-I alpha) from ratbrain, J. Biol. Chem., 269 (45), 28235-28242 (1994)

[0254] 5. Shull, G. E., Greeb, J. and Lingrel, J. B., Molecular cloningof three distinct forms of the Na+, K+-ATPase alpha-subunit from ratbrain, Biochemistry, 25, 8125-8132 (1986).

[0255] 6. Laitala-Leinonen, T., Howell, M. L., Dean, G. E. and Vaananen,H. K., Resorption-cycle-dependent polarization of mRNAs for differentsubunits of V-ATPase in bone-resorbing osteoclasts, Mol. Biol. Cell 7(1), 129-142 (1996).

[0256] 7. Zou, A. P., Yang, Z. Z., Li, P. L. and Cowley A W, J. R.,Oxygen-dependent expression of hypoxia-inducible factor-1alpha in renalmedullary cells of rats, Physiol. Genomics (Online), 6 (3), 159-168(2001).

[0257] 8. Cao, J. L., Revzin, A. and Ferguson-Miller, S., Conversion ofa mitochondrial gene for mammalian cytochrome c oxidase subunit II intoits universal codon equivalent and expression in vivo and in vitro,Biochemistry, 30 (10), 2642-2650 (1991)

[0258] 9. Ji, X., Moore, H. D., Russell, R. G. and Watts, D. J., cDNAcloning and characterization of a rat spermatogenesis-associated proteinRSP29, Biochem. Biophys. Res. Commun., 241 (3), 714-719 (1997).

[0259] 10. Deveraux, Q., Jensen, C. and Rechsteiner, M., Molecularcloning and expression of a 26 S protease subunit enriched in dileucinerepeats, J. Biol. Chem. 270 (40), 23726-23729 (1995); Nomura, N.,Nagase, T., Miyajima, N., Sazuka, T., Tanaka, A., Sato, S., Seki, N.,Kawarabayasi, Y., Ishikawa, K. and Tabata, S., Prediction of the codingsequences of unidentified human genes. II., DNA Res. 1 (5), 223-229(1994).

[0260] 11. Suzuki, T., Nishiyama, K., Yamamoto, A., Inazawa, J., Iwaki,T., Yamada, T., Kanazawa, I. and Sakaki, Y., Molecular cloning of anovel apoptosis-related gene, human nap1 (NCKAP1), and its possiblerelation to alzheimer disease, Genomics, 63 (2), 246-254 (2000).

[0261] 12. Ono, S., Minami, N., Abe, H. and Obinata, T.,Characterization of a novel cofilin isoform which is predominantlyexpressed in mammalian skeletal muscle, J. Biol. Chem., 269, 15280-15286(1994).

[0262] 13. Lafer, E., Zhou, S., Sousa, R. and Tannery, N. H.,Characterization of a novel synapse-specific protein. II. cDNA cloningand sequence analysis of the F1-20 protein, J. Neurosci., 12, 2144-2155(1992)

[0263] 14. Hsueh-Wel Chang et al., UV Inducubility of Rat ProliferatingCell Nuclear Antigen Gene Promoter, J. Cellular Biochem., 73, 423-432(1999) and references cited therein.

[0264] 15. Chan, Y.-L., Lin, A., McNally, J., Peleg, D., Meyuhas, O. andWool, I. G., The primary structure of rat ribosomal protein L19: Adetermination from the sequence of nucleotides in a cDNA and from thesequence of amino acids in the protein, J. Biol. Chem., 262, 1111-1115(1987)

[0265] 16. Watanabe, M., Isobe, T., Okuyama, T., Ichimura, T., Kuwano,R., Takahashi, Y. and Kondo, H., Molecular cloning of cDNA to rat 14-3-3eta chain polypeptide and the neuronal expression of the mRNA in thecentral nervous system, Brain Res. Mol. Brain Res., 10 (2), 151-158(1991); Watanabe, M., Isobe, T., Ichimura, T., Kuwano, R., Takahashi,Y., Kondo, H. and Inoue, Y., Molecular cloning of rat cDNAs for the zetaand theta subtypes of 14-3-3 protein and differential distributions oftheir mRNAs in the brain, Brain Res. Mol. Brain Res., 25 (1-2), 113-121(1994).

1 34 1 604 PRT Rattus norvegicus A-raf protein (AA 1-604) 1 Met Glu ProPro Arg Gly Pro Pro Ala Ser Gly Ala Glu Pro Ser Arg 1 5 10 15 Ala ValGly Thr Val Lys Val Tyr Leu Pro Asn Lys Gln Arg Thr Val 20 25 30 Val ThrVal Arg Asp Gly Met Ser Val Tyr Asp Ser Leu Asp Lys Ala 35 40 45 Leu LysVal Arg Gly Leu Asn Gln Asp Cys Cys Val Val Tyr Arg Leu 50 55 60 Ile LysGly Arg Lys Thr Val Thr Ala Trp Asp Thr Ala Ile Ala Pro 65 70 75 80 LeuAsp Gly Glu Glu Leu Ile Val Glu Val Leu Glu Asp Val Pro Leu 85 90 95 ThrMet His Asn Phe Val Arg Lys Thr Phe Phe Ser Leu Ala Phe Cys 100 105 110Asp Phe Cys Leu Lys Phe Leu Phe His Gly Phe Arg Cys Gln Thr Cys 115 120125 Gly Tyr Lys Phe His Gln His Cys Ser Ser Lys Val Pro Thr Val Cys 130135 140 Val Asp Met Ser Thr Asn Arg Arg Gln Phe Tyr His Ser Ile Gln Asp145 150 155 160 Leu Ser Gly Gly Ser Arg Gln Gln Glu Val Pro Ser Asn LeuSer Val 165 170 175 Asn Glu Leu Leu Thr Pro Gln Gly Pro Ser Pro Phe ThrGln Gln Arg 180 185 190 Asp Gln Glu His Phe Ser Phe Pro Ala Pro Ala AsnPro Pro Leu Gln 195 200 205 Arg Ile Arg Ser Thr Ser Thr Pro Asn Val HisMet Val Ser Thr Thr 210 215 220 Ala Pro Met Asp Ser Ser Leu Met Gln PheThr Ala Gln Ser Phe Ser 225 230 235 240 Thr Asp Ala Ala Gly Arg Gly GlyAsp Gly Ala Pro Arg Gly Ser Pro 245 250 255 Ser Pro Ala Ser Val Ser SerGly Arg Lys Ser Pro His Ser Lys Leu 260 265 270 Pro Ala Glu Gln Arg GluArg Lys Ser Leu Ala Asp Glu Lys Lys Lys 275 280 285 Val Lys Asn Leu GlyTyr Arg Asp Ser Gly Tyr Tyr Trp Glu Val Pro 290 295 300 Pro Ser Glu ValGln Leu Leu Lys Arg Ile Gly Thr Gly Ser Phe Gly 305 310 315 320 Thr ValPhe Arg Gly Arg Trp His Gly Asp Val Ala Val Lys Val Leu 325 330 335 LysVal Ala Gln Pro Thr Ala Glu Gln Ala Gln Ala Phe Lys Asn Glu 340 345 350Met Gln Val Leu Arg Lys Thr Arg His Val Asn Ile Leu Leu Phe Met 355 360365 Gly Phe Met Thr Arg Pro Gly Phe Ala Ile Ile Thr Gln Trp Cys Glu 370375 380 Gly Ser Ser Leu Tyr His His Leu His Val Ala Asp Thr Arg Phe Asp385 390 395 400 Met Val Gln Leu Ile Asp Val Ala Arg Gln Thr Ala Gln GlyMet Asp 405 410 415 Tyr Leu His Ala Lys Asn Ile Ile His Arg Asp Leu LysSer Asn Asn 420 425 430 Ile Phe Leu His Glu Gly Leu Thr Val Lys Ile GlyAsp Phe Gly Leu 435 440 445 Ala Thr Val Lys Thr Arg Trp Ser Gly Ala GlnPro Leu Glu Gln Pro 450 455 460 Ser Gly Ser Val Leu Trp Met Ala Ala GluVal Ile Arg Met Gln Asp 465 470 475 480 Pro Asn Pro Tyr Ser Phe Gln SerAsp Val Tyr Ala Tyr Gly Val Val 485 490 495 Leu Tyr Glu Leu Met Thr GlySer Leu Pro Tyr Ser His Ile Gly Ser 500 505 510 Arg Asp Gln Ile Ile PheMet Val Gly Arg Gly Tyr Leu Ser Pro Asp 515 520 525 Leu Ser Lys Ile PheSer Asn Cys Pro Lys Ala Met Arg Arg Leu Leu 530 535 540 Thr Asp Cys LeuLys Phe Gln Arg Glu Glu Arg Pro Leu Phe Pro Gln 545 550 555 560 Ile LeuAla Thr Ile Glu Leu Leu Gln Arg Ser Leu Pro Lys Ile Glu 565 570 575 ArgSer Ala Ser Glu Pro Ser Leu His Arg Thr Gln Ala Asp Glu Leu 580 585 590Pro Ala Cys Leu Leu Ser Ala Ala Arg Leu Val Pro 595 600 2 2288 DNARattus norvegicus cDNA coding region for A-raf protein 2 gcggtagcgtgtgacaggag gcctatggca cctgcccagt cctacctcag cccatcttga 60 aaaaatccttagacaacatg gaaccaccac gaggccctcc tgctagtggg gctgagccat 120 ctcgggcagttggcactgtc aaagtgtacc tgcctaacaa gcaacgcaca gtggtgactg 180 tccgggatggcatgagtgtc tatgactctt tggacaaggc cctcaaggtg cggggtctca 240 atcaggactgctgtgtggtc tacagactca tcaaaggaag aaagacagtc actgcctggg 300 acacagccattgcacctctg gatggcgagg agctcattgt ggaggtcctg gaagatgtgc 360 cactgaccatgcacaatttt gtacggaaga cattcttcag tttggccttc tgtgacttct 420 gccttaagtttctgttccac ggctttcgct gccaaacctg tggctacaag ttccaccagc 480 attgttcctccaaggtcccc acggtctgcg ttgacatgag taccaaccgc cgacagttct 540 accacagcatccaggatttg tctggaggct ccaggcagca ggaggttccc tcaaatctct 600 ctgtgaatgagctgctaacc ccccagggtc ccagtccctt tacccagcaa cgtgaccagg 660 agcacttctccttccctgcc cctgccaatc ccccactgca gcgcatccgc tccacatcta 720 ctcctaacgtccacatggtc agcaccacag ctcccatgga ctccagcctc atgcagttta 780 ctgctcagagcttcagcacc gatgctgctg gtagaggtgg tgatggagct cctcggggta 840 gccctagcccagctagtgtg tcttcaggga ggaagtcccc acattccaag ttacctgcag 900 aacagcgggaacggaagtcc ttggcagatg aaaagaaaaa agtgaagaac ctggggtacc 960 gggactcaggctattactgg gaggtgccac ccagtgaggt acagctgttg aagaggatcg 1020 ggacaggctcttttggcact gtgtttcggg ggcgttggca tggcgatgta gctgtgaaag 1080 tgctcaaggtggcccaacct accgctgagc aggcccaggc cttcaagaat gagatgcagg 1140 tgctcaggaagacacgacat gtcaacattt tgctgtttat gggtttcatg actcggccag 1200 ggtttgccatcatcacacag tggtgtgagg gttccagcct ctaccaccac ctacatgtgg 1260 ctgacacgcgctttgacatg gtccagctca ttgatgtggc ccggcagact gcccagggca 1320 tggactacctccacgccaag aacatcattc accgagacct aaagtccaac aatatcttcc 1380 tacatgaggggctcacagtc aagattggtg acttcggcct ggccacagtg aagacacgat 1440 ggagtggggcccagccctta gagcagccct cagggtctgt gctgtggatg gcagctgagg 1500 tgatccgaatgcaggacccg aacccctaca gcttccagtc ggatgtctat gcctatggtg 1560 ttgtgctctatgagcttatg accggctcac tgccctacag ccacattggc agccgtgacc 1620 agatcatctttatggtgggt cgtggctatc tgtctccgga cctcagcaaa atcttcagta 1680 attgccccaaagccatgagg cgcttgctga ctgactgcct caagttccag cgggaggagc 1740 ggcctctatttccccagatt ctggccacga tcgagctgct gcagcggtca ctccccaaga 1800 ttgagcggagtgcctccgaa ccctccttgc accgtaccca ggctgatgag ttgcctgcct 1860 gccttctcagcgcagcccgc cttgtgcctt agactccact cccagcccac tagggagcca 1920 ttttcagcttaccatgccaa ggcaccccct tcctaccagc caatcattgt tctgtctgtg 1980 ccctgatactgcctcaggat tccctatccc acaccctggg aaatttgggg gactccaaaa 2040 actgaggtcccctgcttcct ccataatttg gtctcctctt ggctttgggg atagttctaa 2100 tttggagagctgttttacct ccaatggctg ggattcagtg caaagattcc actcggaacc 2160 tctttataaagttttcgcct gacatgtctt cactgaatta tggggttccc agcaccccat 2220 gcggatttgggagtttccct ttgtctcccc ccactattca aggactctcc tctttaccaa 2280 gaagcaca2288 3 327 PRT Rattus norvegicus Protein phosphatase 1, catalyticsubunit 3 Met Ala Asp Gly Glu Leu Asn Val Asp Ser Leu Ile Thr Arg LeuLeu 1 5 10 15 Glu Val Arg Gly Cys Arg Pro Gly Lys Ile Val Gln Met ThrGlu Ala 20 25 30 Glu Val Arg Gly Leu Cys Ile Lys Ser Arg Glu Ile Phe LeuSer Gln 35 40 45 Pro Ile Leu Leu Glu Leu Glu Ala Pro Leu Lys Ile Cys GlyAsp Ile 50 55 60 His Gly Gln Tyr Thr Asp Leu Leu Arg Leu Phe Glu Tyr GlyGly Phe 65 70 75 80 Pro Pro Glu Ala Asn Tyr Leu Phe Leu Gly Asp Tyr ValAsp Arg Gly 85 90 95 Lys Gln Ser Leu Glu Thr Ile Cys Leu Leu Leu Ala TyrLys Ile Lys 100 105 110 Tyr Pro Glu Asn Phe Phe Leu Leu Arg Gly Asn HisGlu Cys Ala Ser 115 120 125 Ile Asn Arg Ile Tyr Gly Phe Tyr Asp Glu CysLys Arg Arg Phe Asn 130 135 140 Ile Lys Leu Trp Lys Thr Phe Thr Asp CysPhe Asn Cys Leu Pro Ile 145 150 155 160 Ala Ala Ile Val Asp Glu Lys IlePhe Cys Cys His Gly Gly Leu Ser 165 170 175 Pro Asp Leu Gln Ser Met GluGln Ile Arg Arg Ile Met Arg Pro Thr 180 185 190 Asp Val Pro Asp Thr GlyLeu Leu Cys Asp Leu Leu Trp Ser Asp Pro 195 200 205 Asp Lys Asp Val GlnGly Trp Gly Glu Asn Asp Arg Gly Val Ser Phe 210 215 220 Thr Phe Gly AlaAsp Val Val Ser Lys Phe Leu Asn Arg His Asp Leu 225 230 235 240 Asp LeuIle Cys Arg Ala His Gln Val Val Glu Asp Gly Tyr Glu Phe 245 250 255 PheAla Lys Arg Gln Leu Val Thr Leu Phe Ser Ala Pro Asn Tyr Cys 260 265 270Gly Glu Phe Asp Asn Ala Gly Gly Met Met Ser Val Asp Glu Thr Leu 275 280285 Met Cys Ser Phe Gln Ile Leu Lys Pro Ser Glu Lys Lys Ala Lys Tyr 290295 300 Gln Tyr Gly Gly Leu Asn Ser Gly Arg Pro Val Thr Pro Pro Arg Thr305 310 315 320 Ala Asn Pro Pro Lys Lys Arg 325 4 2706 DNA Rattusnorvegicus cDNA phosphatase 1, catalytic subunit 4 cgcccttgtt cccgctgcggggaggagagt ctggtgccta caagatggcg gacggggagc 60 tgaacgtgga cagcctcatcacccgcctgc tggaggtacg aggatgtcgt ccgggaaaaa 120 ttgtgcagat gactgaagcagaagtccgag gactgtgtat caagtctcgt gaaatctttc 180 ttagccagcc tattcttttggaattggaag cgccactgaa gatttgtgga gatattcatg 240 gacagtatac agacttactgagattatttg aatatggagg ttttccacca gaagccaact 300 atcttttctt aggagattatgtggacagag gaaagcagtc tttggaaacc atctgtttgc 360 tattggctta caaaatcaaatacccagaga acttctttct tctacgagga aaccatgagt 420 gtgctagcat caaccgcatttatggattct atgatgagtg caaacgaaga tttaatatta 480 aattgtggaa gacattcactgattgtttta attgtctgcc tatagctgct attgttgatg 540 agaaaatctt ctgctgtcatggaggactgt caccagacct acagtctatg gaacagattc 600 ggagaattat gagacccactgacgtacctg atacaggttt gctttgtgat ttactgtggt 660 ccgacccaga taaggatgtacaaggctggg gagaaaatga tcgtggtgtt tcttttactt 720 ttggagctga tgtagtcagtaaatttctga atcgtcatga tttggacttg atttgtcgag 780 cccatcaggt ggtagaagatggatatgaat tttttgctaa acgacaattg gtaactttat 840 tttctgcccc aaattactgcggcgagtttg acaatgctgg tggtatgatg agtgtggatg 900 aaactttgat gtgttcattccagatattga aaccatctga aaagaaagct aagtaccagt 960 atggtgggct gaattctggacgtcctgtca ctccgcctcg aacagctaat ccaccgaaga 1020 aaaggtgaag acaggaattccggaaagaga aaccatcaga tttgttaagg acatacttca 1080 taatatataa gtgtgcactgtaaaaccatc cagccattcg acacccttta tgatgtcaca 1140 cctttaactt aaggagacgggtaaaggatc ttaaattttt ttctaataga aagatgtgct 1200 acactgtatt gtaataagtatactctgtta taatattcaa caaagttaaa tccaaattca 1260 aaagtatcca ttaaagttctatcttctcat atcacagttt ttaaagttga aagcatccca 1320 gttaaactag ctgcgttagttacccagatg agagcatgaa gatccatctg tgtaatgtgg 1380 ctttagtgtt gcttggttgtttctttattt tgggcttgtt ttgttttgtt tgtttttgct 1440 agaataatgg catctacttttcctattttt ccctaaccat tttaaaaagt gaaaatggga 1500 agagctttaa agacattcaccaactattct tttccttcac ttatctactt aaggaactgt 1560 tggatcttac taagaaaacttacgcctcat aataaaaagg aactttagag gccgataggt 1620 tttaaaaata tacaaactatttgatccaat gattttaatc aaacagtttg actgggcaaa 1680 ctttgcagct gataatgactatttcgcttt ttacaaattg ccactgattt ggatttgtgc 1740 actctaacct ttaatttattgatgctctat tgtgcagtag catttcattt aagataaggc 1800 tcatatagta ctatccaaaattagttggta atgtgattat gtggtacctt ggctttaggt 1860 tttaattcgc acgaaacaccttttggcatg cttaactttc tggtattatc ctcacctgca 1920 ttggttttgt tttttggggtttttgttgtt gtttgtttgt ttgtttttag atccacagaa 1980 catgagaatc ctttttgacaagccttggaa agctggctct tctttccctc tctatgtgaa 2040 ggatgtattt aaatgaacactggtcagtgg gacattgtca gctctgagta ttgggtgctt 2100 cactgtctaa taattgccatgtgaatgttg tttttgactg taaggctatg tcactaaaga 2160 tttttactct gcgttttcataatcaaaggt catgatgtgt atagacatgc tttgtagtga 2220 agtatagtag caataatttctgcacatgat caagagttta ttgcagcatt tctttccctg 2280 ttctctcttt tttaagggttagcattaaca aatgtcaagg aatagcaaag tcaacaaaga 2340 ctttaggagg tggaattaagaacacacaga tttgtgatct ttggatgtga cacttattgg 2400 atgttattct aaagtcttattgaacattgt caaatttgta agcttcatgg ggatggacat 2460 aatgtttata taatgcccttcttatgtgtt accatagatg tgaaacctta tattgtcttt 2520 gaaaatgtta aattgagaactctgttaaca ttttatggat tggcacatta tattactgca 2580 agaaacattt gattttcagcacagtgcaaa agttctttaa aatgcatatg tctttttttc 2640 taattcaatt ttgtttaaagcacattttaa atgtagtttt ctcatttagt aaaaagttgt 2700 ctaatt 2706 5 776 PRTRattus norvegicus Protein phosphofructokinase muscle isozyme 5 Met ThrHis Glu Glu His His Glu Ala Lys Thr Leu Gly Ile Gly Lys 1 5 10 15 AlaIle Ala Val Leu Thr Ser Gly Gly Asp Ala Gln Gly Met Asn Ala 20 25 30 ThrVal Arg Ala Val Val Arg Val Gly Ile Phe Thr Gly Leu Arg Val 35 40 45 PhePhe Val His Glu Gly Tyr Gln Gly Leu Val Asp Gly Gly Glu His 50 55 60 IleArg Glu Ala Thr Trp Glu Ser Val Ser Met Met Leu Gln Leu Gly 65 70 75 80Gly Thr Val Ile Gly Ser Ala Arg Cys Lys Asp Phe Arg Glu Arg Glu 85 90 95Gly Arg Leu Arg Ala Ala His Asn Leu Val Lys Arg Gly Ile Thr Asn 100 105110 Leu Cys Val Ile Gly Gly Asp Gly Ser Leu Thr Gly Ala Asp Thr Phe 115120 125 Arg Ser Glu Trp Ser Asp Leu Leu Asn Asp Leu Gln Lys Asp Gly Lys130 135 140 Ile Thr Ala Glu Glu Arg Thr Lys Ser Ser Tyr Leu Asn Ile ValPhe 145 150 155 160 Leu Val Gly Ser Ile Asp Asn Asp Phe Cys Gly Thr AspMet Thr Ile 165 170 175 Gly Thr Asp Ser Ala Leu His Arg Ile Val Glu IleVal Asp Ala Ile 180 185 190 Thr Thr Thr Ala Gln Ser His Gln Arg Thr PheVal Leu Glu Val Met 195 200 205 Gly Arg His Cys Gly Tyr Leu Ala Leu ValThr Ser Leu Ser Cys Gly 210 215 220 Ala Asp Trp Val Phe Ile Pro Glu CysPro Pro Asp Asp Asp Trp Glu 225 230 235 240 Glu His Leu Cys Arg Arg LeuSer Glu Thr Arg Thr Arg Gly Ser Arg 245 250 255 Leu Asn Ile Ile Ile ValAla Glu Gly Ala Ile Asp Lys Asn Gly Lys 260 265 270 Pro Ile Thr Ser GluAsp Ile Lys Asn Leu Val Val Lys Arg Leu Gly 275 280 285 Tyr Asp Thr ArgVal Thr Val Leu Gly His Val Gln Arg Gly Gly Thr 290 295 300 Pro Ser AlaPhe Asp Arg Ile Leu Gly Ser Arg Met Gly Val Glu Ala 305 310 315 320 ValMet Ala Leu Leu Glu Gly Thr Pro Asp Thr Pro Ala Cys Val Val 325 330 335Ser Leu Ser Gly Asn Thr Ala Val Arg Leu Pro Leu Met Glu Cys Val 340 345350 Gln Val Thr Lys Asp Val Thr Lys Ala Met Asp Glu Lys Arg Phe Asp 355360 365 Glu Ala Ile Lys Leu Arg Gly Arg Ser Phe Met Asn Asn Trp Glu Val370 375 380 Tyr Lys Leu Leu Ala His Val Arg Pro Pro Val Ser Lys Gly GlyLeu 385 390 395 400 His Thr Val Ala Val Met Asn Val Gly Ala Pro Ala AlaGly Met Asn 405 410 415 Ala Ala Val Arg Ser Thr Val Arg Ile Gly Leu IleGln Gly Asn Arg 420 425 430 Val Leu Val Val His Asp Gly Phe Glu Gly LeuAla Lys Gly Gln Ile 435 440 445 Glu Glu Ala Gly Trp Ser Tyr Val Gly GlyTrp Thr Gly Gln Gly Gly 450 455 460 Ser Lys Leu Gly Thr Lys Arg Thr LeuPro Lys Lys Asn Leu Glu Gln 465 470 475 480 Ile Ser Ala Asn Ile Thr LysTyr Asn Ile Gln Gly Leu Val Ile Ile 485 490 495 Gly Gly Phe Glu Ala TyrThr Gly Gly Leu Glu Leu Met Glu Gly Arg 500 505 510 Lys Gln Phe Asp GluLeu Cys Ile Pro Phe Val Val Ile Pro Ala Thr 515 520 525 Val Ser Asn AsnVal Pro Gly Ser Asp Phe Ser Ile Gly Ala Asp Thr 530 535 540 Ala Leu AsnThr Ile Cys Thr Thr Cys Asp Arg Ile Lys Gln Ser Ala 545 550 555 560 AlaGly Thr Lys Arg Arg Val Phe Ile Ile Glu Thr Met Gly Gly Tyr 565 570 575Cys Gly Tyr Leu Ala Thr Met Ala Gly Leu Ala Ala Gly Ala Asp Ala 580 585590 Ala Tyr Ile Phe Glu Glu Pro Phe Thr Ile Arg Asp Leu Gln Val Asn 595600 605 Val Glu His Leu Val Gln Lys Met Lys Thr Thr Val Lys Arg Gly Leu610 615 620 Val Leu Arg Asn Glu Lys Cys Asn Glu Asn Tyr Thr Thr Asp PheIle 625 630 635 640 Phe Asn Leu Tyr Ser Glu Glu Gly Lys Gly Ile Phe AspSer Arg Lys 645 650 655 Asn Val Leu Gly His Met Gln Gln Gly Gly Asn ProThr Pro Phe Asp 660 665 670 Arg Asn Phe Ala Thr Lys Met Gly Ala Lys AlaThr Asn Trp Met Ser 675 680 685 Gly Lys Ile Lys Glu Ser Tyr Arg Asn GlyArg Ile Phe Ala Asn Thr 690 695 700 Pro Asp Ser Gly Cys Val Leu Gly MetArg Lys Arg Ala Leu Val Phe 705 710 715 720 Gln Pro Val Thr Glu Leu LysAsp Gln Thr Asp Phe Glu His Arg Ile 725 730 735 Pro Lys Glu Gln Trp TrpLeu Lys Leu Arg Pro Ile Leu Lys Ile Leu 740 745 750 Ala Lys Tyr Glu IleAsp Leu Asp Thr Ser Asp His Ala His Leu Glu 755 760 765 His Ile Ser ArgLys Arg Ser Gly 770 775 6 2757 DNA Rattus norvegicus cDNAphosphofructokinase muscle isozyme 6 gccagcgagg agagctaaaa ctacaagagtggatcatgac ccatgaagag catcatgaag 60 ccaaaaccct ggggatcggc aaggccatcgccgtgttgac ctctggtgga gatgcccaag 120 gtatgaatgc tacggtcagg gctgtggtacgagttggcat cttcaccggg ctccgcgtct 180 tctttgtcca tgagggttac caaggcctggtggatggtgg agagcacatc agggaggcca 240 cctgggagag cgtgtccatg atgctccagctgggaggcac ggtgattgga agtgcccgat 300 gcaaggactt ccgggagcga gaaggacgactccgagctgc ccacaacctg gtgaagcggg 360 ggatcaccaa tctgtgtgtc atcggaggcgatggcagcct cactggggct gacacttttc 420 gttcagagtg gagtgactta ttgaatgacctccagaaaga tgggaagatc acagccgagg 480 agcgtacaaa gtccagctac ctgaacatcgttttcctggt tggctcaatc gacaatgact 540 tctgtggcac tgatatgacc attggtactgactctgccct gcaccgcatt gtagagatcg 600 tggacgccat caccaccacc gctcagagccaccagaggac atttgtgtta gaagtgatgg 660 gccgccactg tggatacctg gcccttgtcacctctctgtc gtgtggggcc gactgggttt 720 tcattcccga gtgtccgcca gatgacgactgggaagaaca cctttgtcgc cgtctcagtg 780 agacaagaac ccgtggttct cgtctcaacatcatcattgt tgctgagggt gcaatcgaca 840 aaaacgggaa gccaatcacc tcagaagacatcaagaacct ggtggtaaag cgtcttggat 900 atgataccag ggtcactgtt ctgggacatgtacagagggg tgggacacca tcagcctttg 960 accggatcct gggcagcagg atgggtgtggaagcagtgat ggcacttttg gaggggaccc 1020 cagacacccc agcctgtgtg gtgagcctctctggtaatac ggctgtgcgc ctgcccctca 1080 tggagtgtgt ccaggtgacc aaagacgtgaccaaggctat ggatgagaag agatttgatg 1140 aagccattaa gctgagaggc cggagcttcatgaacaactg ggaggtatac aagcttctag 1200 ctcatgtcag acccccagtc tctaagggcgggttgcacac ggtggctgtg atgaacgtgg 1260 gggccccagc tgctggaatg aatgctgcagttcgctctac tgtgaggatt ggccttatcc 1320 aaggcaaccg agtgctggtc gtgcatgatggctttgaggg tctggccaaa ggtcagattg 1380 aggaagctgg ctggagctat gttggaggctggactggtca aggtggttcc aaacttggta 1440 ctaaaaggac tctacccaag aagaacctggaacagatcag tgccaacata accaagtata 1500 acatccaggg cctggttatc attgggggctttgaggctta cacagggggc ttggagctga 1560 tggagggcag gaagcagttt gatgagctctgcatcccatt tgtggtcatt cccgccacgg 1620 tttccaataa tgtcccaggg tcagacttcagcatcggggc tgacacagca ctgaacacca 1680 tctgcacgac ctgtgaccga atcaagcagtctgcagcagg caccaagcgg cgagtgttta 1740 tcatcgagac gatgggtggt tactgtggctatctggccac catggcagga ctggcggctg 1800 gggctgatgc tgcctacatt tttgaggagcccttcaccat ccgagatctc caggttaatg 1860 ttgaacatct ggtgcagaag atgaaaacaactgtgaagag aggcctggtg ctgaggaatg 1920 agaagtgcaa cgagaactac actactgatttcattttcaa cctgtactct gaggagggga 1980 agggcatctt cgacagcagg aagaacgtgcttggccacat gcagcagggt ggaaacccaa 2040 ctccctttga caggaatttt gccaccaagatgggtgctaa ggctacgaat tggatgtctg 2100 ggaaaatcaa agagagttac cgtaatggacggatctttgc caacactccc gactcaggtt 2160 gtgttctggg gatgcgtaag agggccctggtctttcagcc agtgactgag ctgaaggacc 2220 agacagattt tgagcaccga atccccaaagaacagtggtg gctgaagctg aggccaatcc 2280 tcaaaatcct ggccaagtac gagattgatctggacacctc tgaccacgcc cacctggagc 2340 acatttccag gaagcggtct ggagaagctgctgtctagac cactttggag tgaggggaaa 2400 atcacctgat catggtcagc tcacaccctggtagacctca gcccatggct tctcagtgtt 2460 gtagcccagc ccctaccctt ctaggtttccctgtactctg tacctgcaac caggatcact 2520 gtggccaggt gttgggggag gggtggtgagtgcctctcct aggtagctgt ccttccttgc 2580 accctggctt catctgtcac acaggctggatgtctctagt gctactgcta gatttcagct 2640 tctcaagagt aaaagtgagc tttatttatttctttgtgat aacaaagagt cctaggtcct 2700 ttccttgtac cacagtgaag tgtaactacactaataaaag ccagctggcc actgtga 2757 7 885 PRT Rattus norvegicus ProteinPhosphodiesterase 1 7 Met Ala Arg Gln Gly Cys Leu Gly Ser Phe Gln ValIle Ser Leu Phe 1 5 10 15 Thr Phe Ala Ile Ser Val Asn Ile Cys Leu GlyPhe Thr Ala Ser Arg 20 25 30 Ile Lys Arg Ala Glu Trp Asp Glu Gly Pro ProThr Val Leu Ser Asp 35 40 45 Ser Pro Trp Thr Asn Thr Ser Gly Ser Cys LysGly Arg Cys Phe Glu 50 55 60 Leu Gln Glu Val Gly Pro Pro Asp Cys Arg CysAsp Asn Leu Cys Lys 65 70 75 80 Ser Tyr Ser Ser Cys Cys His Asp Phe AspGlu Leu Cys Leu Lys Thr 85 90 95 Val Arg Gly Trp Glu Cys Thr Lys Asp ArgSer Gly Glu Val Arg Asn 100 105 110 Glu Glu Asn Ala Cys His Cys Pro GluAsp Cys Leu Ser Arg Gly Asp 115 120 125 Cys Cys Thr Asn Tyr Gln Val ValCys Lys Gly Glu Ser His Trp Val 130 135 140 Asp Asp Ala Ala Arg Asn GlnSer Ser Glu Cys Leu Gln Val Cys Pro 145 150 155 160 Pro Pro Leu Ile IlePhe Ser Val Asp Gly Phe Arg Ala Ser Tyr Met 165 170 175 Lys Lys Gly SerLys Val Met Pro Asn Ile Glu Lys Leu Arg Ser Cys 180 185 190 Gly Thr HisVal Pro Tyr Thr Arg Pro Val Tyr Pro Thr Lys Thr Phe 195 200 205 Pro AsnLeu Tyr Thr Leu Ala Thr Gly Leu Tyr Pro Glu Ser His Gly 210 215 220 IleVal Gly Asn Ser Met Tyr Asp Pro Val Phe Asp Ala Ser Phe His 225 230 235240 Leu Arg Gly Arg Glu Lys Phe Asn His Arg Trp Trp Gly Gly Gln Pro 245250 255 Leu Trp Ile Thr Ala Thr Lys Gln Gly Val Arg Ala Gly Thr Phe Phe260 265 270 Trp Ser Val Ser Ile Pro His Glu Arg Arg Ile Leu Thr Ile LeuGln 275 280 285 Trp Leu Ser Leu Pro Asp Asn Glu Arg Pro Ser Val Tyr AlaPhe Tyr 290 295 300 Ser Glu Gln Pro Asp Phe Ser Gly His Lys Tyr Gly ProPhe Gly Pro 305 310 315 320 Glu Met Thr Asn Pro Leu Arg Glu Ile Asp LysThr Val Gly Gln Leu 325 330 335 Met Asp Gly Leu Lys Gln Leu Arg Leu HisArg Cys Val Asn Val Ile 340 345 350 Phe Val Gly Asp His Gly Met Glu AspVal Thr Cys Asp Arg Thr Glu 355 360 365 Phe Leu Ser Asn Tyr Leu Thr AsnVal Asp Asp Ile Thr Leu Val Pro 370 375 380 Gly Thr Leu Gly Arg Ile ArgAla Lys Ser Ile Asn Asn Ser Lys Tyr 385 390 395 400 Asp Pro Lys Thr IleIle Ala Asn Leu Thr Cys Lys Lys Pro Asp Gln 405 410 415 His Phe Lys ProTyr Met Lys Gln His Leu Pro Lys Arg Leu His Tyr 420 425 430 Ala Asn AsnArg Arg Ile Glu Asp Ile His Leu Leu Val Asp Arg Arg 435 440 445 Trp HisVal Ala Arg Lys Pro Leu Asp Val Tyr Lys Lys Pro Ser Gly 450 455 460 LysCys Phe Phe Gln Gly Asp His Gly Phe Asp Asn Lys Val Asn Ser 465 470 475480 Met Gln Thr Val Phe Val Gly Tyr Gly Pro Thr Phe Lys Tyr Arg Thr 485490 495 Lys Val Pro Pro Phe Glu Asn Ile Glu Leu Tyr Asn Val Met Cys Asp500 505 510 Leu Leu Gly Leu Lys Pro Ala Pro Asn Asn Gly Thr His Gly SerLeu 515 520 525 Asn His Leu Leu Arg Thr Asn Thr Phe Arg Pro Thr Met ProAsp Glu 530 535 540 Val Ser Arg Pro Asn Tyr Pro Gly Ile Met Tyr Leu GlnSer Glu Phe 545 550 555 560 Asp Leu Gly Cys Thr Cys Asp Asp Lys Val GluPro Lys Asn Lys Leu 565 570 575 Glu Glu Leu Asn Lys Arg Leu His Thr LysGly Ser Thr Glu Ala Glu 580 585 590 Thr Gly Lys Phe Arg Gly Ser Lys HisGlu Asn Lys Lys Asn Leu Asn 595 600 605 Gly Ser Val Glu Pro Arg Lys GluArg His Leu Leu Tyr Gly Arg Pro 610 615 620 Ala Val Leu Tyr Arg Thr SerTyr Asp Ile Leu Tyr His Thr Asp Phe 625 630 635 640 Glu Ser Gly Tyr SerGlu Ile Phe Leu Met Pro Leu Trp Thr Ser Tyr 645 650 655 Thr Ile Ser LysGln Ala Glu Val Ser Ser Ile Pro Glu His Leu Thr 660 665 670 Asn Cys ValArg Pro Asp Val Arg Val Ser Pro Gly Phe Ser Gln Asn 675 680 685 Cys LeuAla Tyr Lys Asn Asp Lys Gln Met Ser Tyr Gly Phe Leu Phe 690 695 700 ProPro Tyr Leu Ser Ser Ser Pro Glu Ala Lys Tyr Asp Ala Phe Leu 705 710 715720 Val Thr Asn Met Val Pro Met Tyr Pro Ala Phe Lys Arg Val Trp Ala 725730 735 Tyr Phe Gln Arg Val Leu Val Lys Lys Tyr Ala Ser Glu Arg Asn Gly740 745 750 Val Asn Val Ile Ser Gly Pro Ile Phe Asp Tyr Asn Tyr Asp GlyLeu 755 760 765 Arg Asp Thr Glu Asp Glu Ile Lys Gln Tyr Val Glu Gly SerSer Ile 770 775 780 Pro Val Pro Thr His Tyr Tyr Ser Ile Ile Thr Ser CysLeu Asp Phe 785 790 795 800 Thr Gln Pro Ala Asp Lys Cys Asp Gly Pro LeuSer Val Ser Ser Phe 805 810 815 Ile Leu Pro His Arg Pro Asp Asn Asp GluSer Cys Asn Ser Ser Glu 820 825 830 Asp Glu Ser Lys Trp Val Glu Glu LeuMet Lys Met His Thr Ala Arg 835 840 845 Val Arg Asp Ile Glu His Leu ThrGly Leu Asp Phe Tyr Arg Lys Thr 850 855 860 Ser Arg Ser Tyr Ser Glu IleLeu Thr Leu Lys Thr Tyr Leu His Thr 865 870 875 880 Tyr Glu Ser Glu Ile885 8 3216 DNA Rattus norvegicus cDNA Phosphodiesterase 1 8 ggtacccaacagcctgaact cagagccccg agagcagagc attcagggca agcagaaaca 60 ccctgcagaggctttccaag aatccctcgg catggcaaga caaggctgtc tcgggtcatt 120 ccaggtaatatccttgttca cttttgccat cagtgtcaat atctgcttag gattcacagc 180 aagtcgaattaagagggcag aatgggatga aggacctccc acagtgctgt ctgactctcc 240 atggaccaacacctctggat cctgcaaagg tagatgcttt gagcttcaag aggttggccc 300 tccagactgtcggtgtgaca acctgtgtaa gagctacagc agctgctgcc acgatttcga 360 tgagctctgtttgaaaacag tccgaggctg ggagtgcacc aaagacagaa gtggggaagt 420 acgaaacgaggaaaatgcct gtcactgccc agaagactgc ttgtccaggg gagactgctg 480 taccaactaccaagtggtct gcaaaggaga atcacactgg gtagatgatg ctgcgagaaa 540 tcaaagttccgaatgcctgc aggtttgtcc gcctccgtta atcatcttct ctgtggatgg 600 tttccgtgcatcatacatga agaaaggcag caaggttatg cccaacattg agaaactgcg 660 gtcctgtggcacccatgtcc cctacacgag gcctgtgtac cccacaaaaa ccttccctaa 720 tctatatacgctggccactg gtttatatcc ggaatcccat ggaattgtcg gtaattcaat 780 gtatgatcctgtctttgatg cttcgttcca tctacgaggg cgagagaagt ttaatcatag 840 gtggtggggaggccaaccgc tatggattac agccaccaag caaggggtga gagctggaac 900 attcttttggtctgtgagca tccctcatga acggaggatc ctaaccattc ttcagtggct 960 ttctctgccagacaacgaga ggccttcagt ttatgccttc tactcagagc agcctgattt 1020 ttctggacacaagtacggcc cttttggccc tgagatgaca aatcctctga gggagattga 1080 caagaccgtggggcagttaa tggatggact gaaacaactc aggctgcatc gctgtgtgaa 1140 cgttatctttgttggagacc atggaatgga agatgtgaca tgtgacagaa ctgagttctt 1200 gagcaactatctgactaatg tggatgacat tactttagtg cctggaactc tgggaagaat 1260 tcgagccaaatctatcaata attctaaata tgaccctaaa accattattg ctaacctcac 1320 gtgcaaaaaaccggatcagc actttaagcc ttacatgaaa cagcaccttc ccaaacggtt 1380 gcactatgccaacaacagaa gaattgaaga catccattta ttggtcgatc gaagatggca 1440 tgttgcaaggaaacctttgg acgtttataa gaaaccatca ggaaaatgtt ttttccaggg 1500 tgaccacggctttgataaca aggtcaatag catgcagact gttttcgtag gttatggccc 1560 aacttttaagtacaggacta aagtgcctcc atttgaaaac attgaacttt acaatgttat 1620 gtgcgatctcctaggcttga agcccgctcc caataatgga actcatggaa gcttgaatca 1680 cctactgcgtacaaatacct ttaggccaac catgccagac gaagtcagcc gacctaacta 1740 cccagggattatgtaccttc agtccgagtt tgacctgggc tgcacctgtg acgataaggt 1800 agagccaaagaacaaattgg aagaactcaa taaacgtctt cataccaaag gatcaacaga 1860 agctgaaaccgggaaattca gaggcagcaa acatgaaaac aagaaaaacc ttaatggaag 1920 tgttgaacctagaaaagaga gacatctcct gtatggacgg cctgcagtgc tctatcggac 1980 tagctatgatatcttatacc atacggactt tgaaagtggt tatagtgaaa tattcttaat 2040 gcctctctggacatcgtata ccatttctaa gcaggctgag gtctccagca tcccagaaca 2100 cctgaccaactgtgttcgtc ctgatgtccg tgtgtctcca ggattcagtc agaactgttt 2160 agcttataaaaatgataaac agatgtcata tggattcctt tttcctccct acctgagctc 2220 ctccccagaagctaagtatg atgcattcct cgtaaccaac atggttccaa tgtaccccgc 2280 cttcaaacgtgtttgggctt atttccaaag ggttttggtg aagaaatatg cttcagaaag 2340 gaatggagtcaacgtaataa gtggaccgat ttttgactac aattacgatg gcctacgtga 2400 cactgaagatgaaattaaac agtatgtgga aggcagctct atacctgtcc ccacccacta 2460 ctacagcatcatcaccagct gcctggactt cactcagcct gcagacaagt gtgacggtcc 2520 cctctctgtgtcttccttca tccttcctca ccgacccgac aatgatgaga gctgtaatag 2580 ctccgaggatgagtcgaagt gggtagagga actcatgaag atgcacacag ctcgggtgcg 2640 ggacattgagcacctcactg gtctggattt ctaccggaag actagccgta gctattcgga 2700 aattctgaccctcaagacat acctgcatac atatgagagc gagatttaac tttctgggcc 2760 tgggcagtgtagtcttagca actggtgtat atttttatat tgtgtttgta tttattaatt 2820 tgaaccaggacacaaacaaa caaagaaaca aacaaataaa aaaaaaaacc acttagtatt 2880 ttaatcctgtaccaaatctg acatattaag ctgaatgact gtgctatttt ttttccttaa 2940 ttcttgatttagacagagtt gtgttctgag cagagtttat agtgaacact gaggctcaca 3000 atccaagtagaagctacgtg gatctacaag gtgctgcagg ttgaaaattt gcattgagga 3060 aatattagttttccagggca cagtcaccac gtgtagttct gttctgtttt gaaagactga 3120 ttttgtaaaggtgcattcat ctgctgttaa ctttgacaga catatttatg ccttatagac 3180 caagcttaaatataataaat cacacattca gatttt 3216 9 1020 PRT Rattus norvegicus ProteinNa+, K+ ATPase alpha(+) isoform catalytic subunit 9 Met Gly Arg Gly AlaGly Arg Glu Tyr Ser Pro Ala Ala Thr Thr Ala 1 5 10 15 Glu Asn Gly GlyGly Lys Lys Lys Gln Lys Glu Lys Glu Leu Asp Glu 20 25 30 Leu Lys Lys GluVal Ala Met Asp Asp His Lys Leu Ser Leu Asp Glu 35 40 45 Leu Gly Arg LysTyr Gln Val Asp Leu Ser Lys Gly Leu Thr Asn Gln 50 55 60 Arg Ala Gln AspIle Leu Ala Arg Asp Gly Pro Asn Ala Leu Thr Pro 65 70 75 80 Pro Pro ThrThr Pro Glu Trp Val Lys Phe Cys Arg Gln Leu Phe Gly 85 90 95 Gly Phe SerIle Leu Leu Trp Ile Gly Ala Leu Leu Cys Phe Leu Ala 100 105 110 Tyr GlyIle Leu Ala Ala Met Glu Asp Glu Pro Ser Asn Asp Asn Leu 115 120 125 TyrLeu Gly Ile Val Leu Ala Ala Val Val Ile Val Thr Gly Cys Phe 130 135 140Ser Tyr Tyr Gln Glu Ala Lys Ser Ser Lys Ile Met Asp Ser Phe Lys 145 150155 160 Asn Met Val Pro Gln Gln Ala Leu Val Ile Arg Glu Gly Glu Lys Met165 170 175 Gln Ile Asn Ala Glu Glu Val Val Val Gly Asp Leu Val Glu ValLys 180 185 190 Gly Gly Asp Arg Val Pro Ala Asp Leu Arg Ile Ile Ser SerHis Gly 195 200 205 Cys Lys Val Asp Asn Ser Ser Leu Thr Gly Glu Ser GluPro Gln Thr 210 215 220 Arg Ser Pro Glu Phe Thr His Glu Asn Pro Leu GluThr Arg Asn Ile 225 230 235 240 Cys Phe Phe Ser Thr Asn Cys Val Glu GlyThr Ala Arg Gly Ile Val 245 250 255 Ile Ala Thr Gly Asp Arg Thr Val MetGly Arg Ile Ala Thr Leu Ala 260 265 270 Ser Gly Leu Glu Val Gly Gln ThrPro Ile Ala Met Glu Ile Glu His 275 280 285 Phe Ile Gln Leu Ile Thr GlyVal Ala Val Phe Leu Gly Val Ser Phe 290 295 300 Phe Val Leu Ser Leu IleLeu Gly Tyr Ser Trp Leu Glu Ala Val Ile 305 310 315 320 Phe Leu Ile GlyIle Ile Val Ala Asn Val Pro Glu Gly Leu Leu Ala 325 330 335 Thr Val ThrVal Cys Leu Thr Leu Thr Ala Lys Arg Met Ala Arg Lys 340 345 350 Asn CysLeu Val Lys Asn Leu Glu Ala Val Glu Thr Leu Gly Ser Thr 355 360 365 SerThr Ile Cys Ser Asp Lys Thr Gly Thr Leu Thr Gln Asn Arg Met 370 375 380Thr Val Ala His Met Trp Phe Asp Asn Gln Ile His Glu Ala Asp Thr 385 390395 400 Thr Glu Asp Gln Ser Gly Ala Thr Phe Asp Lys Arg Ser Pro Thr Trp405 410 415 Thr Ala Leu Ser Arg Ile Ala Gly Leu Cys Asn Arg Ala Val PheLys 420 425 430 Ala Gly Gln Glu Asn Ile Ser Val Ser Lys Arg Asp Thr AlaGly Asp 435 440 445 Ala Ser Glu Ser Ala Leu Leu Lys Cys Ile Glu Leu SerCys Gly Ser 450 455 460 Val Arg Lys Met Arg Asp Arg Asn Pro Lys Val AlaGlu Ile Pro Phe 465 470 475 480 Asn Ser Thr Asn Lys Tyr Gln Leu Ser IleHis Glu Arg Glu Asp Ser 485 490 495 Pro Gln Ser His Val Leu Val Met LysGly Ala Pro Glu Arg Ile Leu 500 505 510 Asp Arg Cys Ser Thr Ile Leu ValGln Gly Lys Glu Ile Pro Leu Asp 515 520 525 Lys Glu Met Gln Asp Ala PheGln Asn Ala Tyr Met Glu Leu Gly Gly 530 535 540 Leu Gly Glu Arg Val LeuGly Phe Cys Gln Leu Asn Leu Pro Ser Gly 545 550 555 560 Lys Phe Pro ArgGly Phe Lys Phe Asp Thr Asp Glu Leu Asn Phe Pro 565 570 575 Thr Glu LysLeu Cys Phe Val Gly Leu Met Ser Met Ile Asp Pro Pro 580 585 590 Arg AlaAla Val Pro Asp Ala Val Gly Lys Cys Arg Ser Ala Gly Ile 595 600 605 LysVal Ile Met Val Thr Gly Asp His Pro Ile Thr Ala Lys Ala Ile 610 615 620Ala Lys Gly Val Gly Ile Ile Ser Glu Gly Asn Glu Thr Val Glu Asp 625 630635 640 Ile Ala Ala Arg Leu Asn Ile Pro Val Ser Gln Val Asn Pro Arg Glu645 650 655 Ala Lys Ala Cys Val Val His Gly Ser Asp Leu Lys Asp Met ThrSer 660 665 670 Glu Gln Leu Asp Glu Ile Leu Arg Asp His Thr Glu Ile ValPhe Ala 675 680 685 Arg Thr Ser Pro Gln Gln Lys Leu Ile Ile Val Glu GlyCys Gln Arg 690 695 700 Gln Gly Ala Ile Val Ala Val Thr Gly Asp Gly ValAsn Asp Ser Pro 705 710 715 720 Ala Leu Lys Lys Ala Asp Ile Gly Ile AlaMet Gly Ile Ser Gly Ser 725 730 735 Asp Val Ser Lys Gln Ala Ala Asp MetIle Leu Leu Asp Asp Asn Phe 740 745 750 Ala Ser Ile Val Thr Gly Val GluGlu Gly Arg Leu Ile Phe Asp Asn 755 760 765 Leu Lys Lys Ser Ile Ala TyrThr Leu Thr Ser Asn Ile Pro Glu Ile 770 775 780 Thr Pro Phe Leu Leu PheIle Ile Ala Asn Ile Pro Leu Pro Leu Gly 785 790 795 800 Thr Val Thr IleLeu Cys Ile Asp Leu Gly Thr Asp Met Val Pro Ala 805 810 815 Ile Ser LeuAla Tyr Glu Ala Ala Glu Ser Asp Ile Met Lys Arg Gln 820 825 830 Pro ArgAsn Ser Gln Thr Asp Lys Leu Val Asn Glu Arg Leu Ile Ser 835 840 845 MetAla Tyr Gly Gln Ile Gly Met Ile Gln Ala Leu Gly Gly Phe Phe 850 855 860Thr Tyr Phe Val Ile Leu Ala Glu Asn Gly Phe Leu Pro Ser Arg Leu 865 870875 880 Leu Gly Ile Arg Leu Asp Trp Asp Asp Arg Thr Thr Asn Asp Leu Glu885 890 895 Asp Ser Tyr Gly Gln Glu Trp Thr Tyr Glu Gln Arg Lys Val ValGlu 900 905 910 Phe Thr Cys His Thr Ala Phe Phe Ala Ser Ile Val Val ValGln Trp 915 920 925 Ala Asp Leu Ile Ile Cys Lys Thr Arg Arg Asn Ser ValPhe Gln Gln 930 935 940 Gly Met Lys Asn Lys Ile Leu Ile Phe Gly Leu LeuGlu Glu Thr Ala 945 950 955 960 Leu Ala Ala Phe Leu Ser Tyr Cys Pro GlyMet Gly Val Ala Leu Arg 965 970 975 Met Tyr Pro Leu Lys Val Thr Trp TrpPhe Cys Ala Phe Pro Tyr Ser 980 985 990 Leu Leu Ile Phe Ile Tyr Asp GluVal Arg Lys Leu Ile Leu Arg Arg 995 1000 1005 Tyr Pro Gly Gly Trp ValGlu Lys Glu Thr Tyr Tyr 1010 1015 1020 10 5109 DNA Rattus norvegicuscDNA Na+, K+ ATPase alpha(+) isoform catalytic subunit 10 tctgccagggtctccagctg ccccagacag gcggtgtggt cttgggatcc tcctggtgac 60 ctttccagcctaggtcccct cagccactct gccccaagat gggacgtggg gcagggcgtg 120 agtactcgcctgccgccacc actgcggaga atgggggtgg caagaagaaa cagaaagaga 180 aggagctcgatgagctgaag aaggaggttg ccatggatga ccacaagctg tccttggatg 240 agctgggccgaaaataccaa gtggatctgt ccaagggcct caccaaccag cgagctcagg 300 atattctggctagagacgga cccaacgccc tcactccacc ccctacaact cctgagtggg 360 tcaagttctgccgtcagctt tttgggggct tctctatcct gctgtggatt ggggcgcttc 420 tctgcttcttagcctatggt atcctggccg ccatggagga cgaaccatcc aatgacaatt 480 tatatctaggtatcgtgcta gcagctgtag ttatcgtcac tggctgcttc tcctactacc 540 aggaagccaaaagctccaag attatggact ccttcaagaa catggtgcct cagcaagctc 600 tggtcatccgagagggagag aagatgcaga tcaatgcaga ggaggtggtc gtgggagacc 660 tggtggaagtgaagggtgga gaccgtgtcc ccgctgacct ccggatcatc tcctcccacg 720 gttgcaaggtggataactca tccctgacag gggagtcgga gccccagacc cggtcccctg 780 agttcacccatgagaatccc ttggagaccc gcaatatctg tttcttctct accaactgtg 840 tggaaggcactgccaggggc attgtgatcg ccacaggtga ccggacggtg atgggccgca 900 tagccactcttgcctctggc ctagaggtgg gacagacgcc gatagccatg gagatcgagc 960 atttcatccagctgatcacg ggggtggccg tgttcctggg ggtctccttc tttgttctgt 1020 cgctcatcctgggctacagc tggctggagg ctgtcatctt cctcatcggc atcatcgtag 1080 ccaacgtccccgaagggctc ttggccactg ttactgtgtg cctgacgctg acagccaagc 1140 gcatggctcgcaagaactgc ctggtgaaga acctggaggc ggtggagacg ctgggctcca 1200 cgtccaccatctgctcggac aagacaggca ccctcaccca gaaccgcatg acggtggctc 1260 acatgtggtttgacaaccag atccatgagg ctgacaccac tgaagatcag tctggggcca 1320 cttttgacaagcggtccccg acgtggacag ccctgtctcg gatcgctggt ctctgcaatc 1380 gtgccgtcttcaaggctggg caggagaaca tctccgtgtc taagcgggac acagctggtg 1440 acgcctctgagtcagctctg ctcaagtgca tcgagttgtc ctgtggctca gtgaggaaga 1500 tgagggacaggaatcccaag gtggcagaaa ttcccttcaa ctctaccaac aaatatcagc 1560 tttccatccatgagagggaa gacagccccc agagccatgt gctggtgatg aaaggtgccc 1620 cggagcgcatcctggaccga tgctctacca tcctggtaca gggcaaggag atccctcttg 1680 acaaggagatgcaagatgcc tttcaaaacg cctacatgga gctgggagga ctcggggagc 1740 gagtgctgggcttctgtcag ctgaacctgc cttctggaaa gtttcctcgg ggcttcaaat 1800 ttgacacggatgagctgaac tttcccacag agaagctctg ctttgtgggg ctcatgtcta 1860 tgattgatccccccagagca gctgtgccag atgctgtggg caagtgcaga agtgcaggca 1920 tcaaggtgatcatggtgact ggggatcacc ctatcacagc caaggccatt gccaaaggtg 1980 tgggcatcatatcagagggt aacgagactg tggaagacat tgcagccagg ctcaacattc 2040 ctgtgagtcaagtcaatccc agagaagcca aggcatgtgt agtgcacggc tcagacctga 2100 aggacatgacttcagagcag ctggatgaga tcctcaggga ccacacggag atcgtgtttg 2160 cccggacctcccctcagcag aagctcatca ttgtggaggg ctgtcagagg cagggagcca 2220 tcgtggcagtgactggtgac ggggtgaacg actcccccgc gctgaagaag gctgacattg 2280 gcattgccatgggcatctct ggctctgatg tctctaagca ggcagctgac atgatccttc 2340 tcgacgacaactttgcctcc attgtgacgg gcgtggagga ggggcgcctg atctttgaca 2400 acctgaagaagtccatcgcg tacaccctga ccagcaacat ccctgagatc acccccttcc 2460 tgctgttcatcattgccaac atcccccttc ccctgggcac cgtgaccatc ctgtgcatcg 2520 acctgggcacagacatggtt cctgccatct cattagcata cgaagcggct gagagcgaca 2580 tcatgaagaggcagccacgg aactcccaga cggacaagct ggtgaacgag aggcttatca 2640 gcatggcttacggacagatc ggcatgatcc aggctctggg cggcttcttc acctactttg 2700 taatactggcagagaacggc ttcctgccat cgaggctgct tgggatccgc cttgactggg 2760 atgatcggactaccaacgac ctggaggaca gctatggaca agagtggacc tatgagcagc 2820 ggaaggtggtggagttcaca tgccacacgg ccttctttgc cagcatcgtg gttgtgcagt 2880 gggctgacctcatcatttgc aagacccggc gcaactcggt gttccagcag ggcatgaaga 2940 acaagatcctgatttttggg ctgctagaag agacggctct ggctgccttc ctgtcttact 3000 gcccgggtatgggggtggcc ctccgaatgt acccactcaa ggtcacgtgg tggttctgtg 3060 cctttccctacagtctcctc atcttcatct atgatgaagt ccgaaagctc atcctgcggc 3120 ggtaccctgggggctgggtg gagaaggaga cgtactactg agctcaccga caaaaggaag 3180 aacaggggagatggggtgct ccagaggggc tggtgggtgt tgtggtgaag ggaagggctg 3240 gggagacacaaggaagcgat ggtggcgtga actcagtggg taggcttggg taaataaact 3300 tgaggagactgctccaactg ctccataggt cccgctgtga accctaagac agtgcatgtt 3360 ggggtcgcctcctcagatcc ttcccgtccc actctcccac attgtctaca ttttctgaag 3420 aaccgggggtcgccctagcc ctccctgtgt cccagtcctt caccctcacc tgcattattc 3480 cattattcaaacagatcaac acccaaaggt taatcctgtc taaccctgga ggaaagcctg 3540 tcagaccaccagtacccacc actaccacca ccaccaccac caccaccacc accaccacca 3600 ccaccaccaccaccccctgt tcactcctct tcccattctt gccttcctca ccttcctgcc 3660 tgagtcttcccttgctcctc cccttacacc ttgaaaacac aaaattctgc ttctgtgagt 3720 gcaagagcctagggccagaa aaggaagcca gttggagaga tggggcctgt ctcccagcca 3780 agaccagtcaggaaccagag gggagctggg ctggcaagtg gaggttgggg tgcactggct 3840 gagaaagaaagaaaggaagg aaggaaggaa ggaaggaagg aaggaagaaa ggaaggaagg 3900 aagtgaccacaggtgtgtca tctccagcct tcaggtatat gggacaggct ctggatctgt 3960 gagagacttaagagactagc acaccagcag accaaattcc catctcatca gactagcagt 4020 aagtgccacccagtgcccct ctgacccttg ggtagtggtc ctctctgtcc acaaggctca 4080 gatttcacagaaggttcagc tatctcaacc acatactctt gggaacaccc cccttcttta 4140 gaataattagttctctgggg cctcgtgctg ttctgagagc cccattagct gccacttctc 4200 ctcgtgctctctcactgcct tctgcttcct acccacctgc tgaacccacg ttatgtccag 4260 tatcgccttgcttgtcctga aaaaggatct cttggccatt ggcaggaatc agtgtagaaa 4320 tgtttccaggacatccctga ctttcgggaa catgcagaat cagtgtagct catgacacag 4380 tcagaaactttagacacaag agaaattctt aagagaccta tgcacctttg acctctcaga 4440 ttgagacaggaagctggctt caggttccta tggcatagag ttatctttcc ttactctctt 4500 ctcaacctaacgtattcgct cttcagacag ctgcctgttc tctaatcctg gcctagaaag 4560 catagcatagatgcacctgg atcaatgagg gaagcaagag agaatcagca aggaaactgg 4620 aaggcttgaggtgggaatat gaaagtcaag acaagcatca agccaggccc aagggcctcc 4680 caaaggctactgtctcatcg tggtggatgg agttttgcct taccctaaat accttgaaat 4740 ttgtcagtcatgcatagcct tcagtcggaa gtcaaaatgg gacaccgtat ttatttgggc 4800 ctgactaatttgagatcact gactttgaac aaaagtttac ctttgcacaa tcaataaaat 4860 catctgctaggtaattcaag agcataaacg atactgctag gagcagcata gttagtttca 4920 aagtatgctttccgagcact ttagcaatct ccctttagaa tcaggaagtg cataggctaa 4980 ttactatcagtcccgatata tttgttaaag gaacacctac aagatcctta ctggtgacct 5040 tctgtgagacactagtttga ggcactacat gtgtacttga aaataataaa gttgcatttc 5100 tttatgaat5109 11 617 PRT Mus musculus Protein vacuolar adenosine triphosphatasesubunit A 11 Met Asp Phe Ser Lys Leu Pro Lys Ile Arg Asp Glu Asp Lys GluSer 1 5 10 15 Thr Phe Gly Tyr Val His Gly Val Ser Gly Pro Val Val ThrAla Cys 20 25 30 Asp Met Ala Gly Ala Ala Met Tyr Glu Leu Val Arg Val GlyHis Ser 35 40 45 Glu Leu Val Gly Glu Ile Ile Arg Leu Glu Gly Asp Met AlaThr Ile 50 55 60 Gln Val Tyr Glu Glu Thr Ser Gly Val Ser Val Gly Asp ProVal Leu 65 70 75 80 Arg Thr Gly Lys Pro Arg Ser Val Glu Leu Gly Pro GlyIle Met Gly 85 90 95 Ala Ile Phe Asp Gly Ile Gln Arg Pro Leu Ser Asp IleSer Ser Gln 100 105 110 Thr Gln Ser Ile Tyr Ile Pro Arg Gly Val Asn ValSer Ala Leu Ser 115 120 125 Arg Asp Ile Lys Trp Glu Phe Ile Pro Ser LysAsn Leu Arg Val Gly 130 135 140 Ser His Ile Thr Gly Gly Asp Ile Tyr GlyIle Val Asn Glu Asn Ser 145 150 155 160 Leu Ile Lys His Lys Ile Met LeuPro Pro Arg Asn Arg Gly Ser Val 165 170 175 Thr Tyr Ile Ala Pro Pro GlyAsn Tyr Asp Ala Ser Asn Val Val Leu 180 185 190 Glu Leu Glu Phe Glu GlyVal Lys Glu Lys Phe Ser Met Val Gln Val 195 200 205 Trp Pro Val Arg GlnVal Arg Pro Val Thr Glu Lys Leu Pro Ala Asn 210 215 220 His Pro Leu LeuThr Gly Gln Arg Val Leu Asp Ala Leu Phe Pro Cys 225 230 235 240 Val GlnGly Gly Thr Thr Ala Ile Pro Gly Ala Phe Gly Cys Gly Lys 245 250 255 ThrVal Ile Ser Gln Ser Leu Ser Lys Tyr Ser Asn Ser Asp Val Ile 260 265 270Ile Tyr Val Gly Cys Gly Glu Arg Gly Asn Glu Met Ser Glu Val Leu 275 280285 Arg Asp Phe Pro Glu Leu Thr Met Glu Val Asp Gly Lys Ala Glu Ser 290295 300 Ile Met Lys Arg Thr Ala Leu Val Ala Asn Thr Ser Asn Met Pro Val305 310 315 320 Ala Ala Arg Glu Ala Ser Ile Tyr Thr Gly Ile Thr Leu SerGlu Tyr 325 330 335 Phe Arg Asp Met Gly Tyr His Val Ser Met Met Ala AspSer Thr Ser 340 345 350 Arg Trp Ala Glu Ala Leu Arg Glu Ile Ser Gly ArgLeu Ala Glu Met 355 360 365 Pro Ala Asp Ser Gly Tyr Pro Ala Tyr Leu GlyAla Arg Leu Ala Ser 370 375 380 Phe Tyr Glu Arg Ala Gly Arg Val Lys CysLeu Gly Asn Pro Glu Arg 385 390 395 400 Glu Gly Ser Val Ser Ile Val GlyAla Val Ser Pro Pro Gly Gly Asp 405 410 415 Phe Ser Asp Pro Val Thr SerAla Thr Leu Gly Ile Val Gln Val Phe 420 425 430 Trp Gly Leu Asp Lys LysLeu Ala Gln Arg Lys His Phe Pro Ser Val 435 440 445 Asn Trp Leu Ile SerTyr Ser Lys Tyr Met Arg Ala Leu Asp Glu Tyr 450 455 460 Tyr Asp Lys HisPhe Thr Glu Phe Val Pro Leu Arg Thr Lys Ala Lys 465 470 475 480 Glu IleLeu Gln Glu Glu Gly Asp Leu Ala Glu Ile Val Gln Leu Val 485 490 495 GlyLys Ala Ser Leu Ala Glu Thr Asp Lys Ile Thr Leu Glu Val Ala 500 505 510Lys Leu Ile Lys Asp Asp Phe Leu Gln Gln Asn Gly Tyr Thr Pro Tyr 515 520525 Asp Arg Phe Cys Pro Phe Tyr Lys Thr Val Gly Met Leu Ser Asn Met 530535 540 Ile Ser Phe Tyr Asp Met Ala Arg Arg Ala Val Glu Thr Thr Ala Gln545 550 555 560 Ser Asp Asn Lys Ile Thr Trp Ser Ile Ile Arg Glu His MetGly Glu 565 570 575 Ile Leu Tyr Lys Leu Ser Ser Met Lys Phe Lys Asp ProVal Lys Asp 580 585 590 Gly Glu Ala Lys Ile Lys Ala Asp Tyr Ala Gln LeuLeu Glu Asp Met 595 600 605 Gln Asn Ala Phe Arg Ser Leu Glu Asp 610 61512 3007 DNA Mus musculus cDNA vacuolar adenosine triphosphatase subunitA 12 gcaggtaaat ttaacacaat ggatttctcc aagctaccca aaatccgaga tgaggataaa60 gaaagtacat ttggttatgt gcatggagtc tcagggcctg tggttacagc ctgtgacatg 120gcgggcgctg ccatgtacga gctggtgaga gtggggcaca gcgagctggt tggagaaatt 180attcgattgg aaggtgacat ggccaccatt caggtgtatg aagaaacttc tggtgtgtct 240gttggagacc ccgtactccg cactggtaaa cctcgctcgg tcgagctggg tcccgggatt 300atgggagcca tttttgatgg tatacagaga cctctgtcgg atatcagcag tcagacccaa 360agtatctaca tccccagagg agtcaatgtg tctgctctca gcagagatat caaatgggag 420tttataccca gcaaaaacct acgggttggt agtcatatca ctggtggaga catttatggg 480attgtcaatg agaactccct catcaaacac aaaatcatgt tgcccccacg taacagagga 540agcgtgactt acatcgcgcc gcctgggaat tatgatgcat cgaatgtcgt cctggagctt 600gagtttgaag gtgtgaagga gaagttcagc atggtccaag tgtggcctgt gcggcaggtg 660cggcctgtca ctgagaagct gcccgccaat caccccttac ttactggcca gagagtcctc 720gatgcccttt tcccgtgtgt tcagggagga actactgcta tcccaggcgc ctttggctgt 780ggaaagactg tgatttccca gtctctatcc aagtactcca acagtgacgt catcatctat 840gtcggctgcg gtgagagagg caacgagatg tcagaagttc tccgagactt ccctgagctc 900accatggagg ttgatgggaa agcagagtcc atcatgaaga ggacagcgct ggtagccaac 960acctccaaca tgcctgtggc tgcgagagag gcctccatct acactggaat tacactatca 1020gaatatttcc gtgacatggg ctaccacgtc agtatgatgg ccgactctac ctctagatgg 1080gctgaggccc tcagagaaat ctctggtcgt ttagctgaga tgcctgcaga tagtggatac 1140cctgcatacc ttggtgcccg gctggcttct ttctatgagc gagcaggcag agtgaaatgt 1200ctcggaaacc ctgagagaga agggagtgtc agcattgtag gagcagtttc tccacctggt 1260ggtgattttt ctgatccagt cacttctgca acgctgggta ttgttcaggt gttctggggc 1320ttggataaga agttagctca gcgcaagcac ttcccctctg tcaactggct catcagctac 1380agcaagtaca tgcgtgccct ggacgagtac tatgacaagc acttcacgga gtttgttcct 1440ctgaggacca aagctaagga gatcctgcag gaagaagggg atctggcaga aatcgtgcag 1500ctcgtgggca aggcctcttt agcagagacg gataaaatca ctctggaggt agcaaaactt 1560attaaagatg acttcctaca gcaaaatggg tacactcctt atgacaggtt ctgtccattc 1620tacaagacag tggggatgct gtccaacatg atttcattct atgacatggc ccgccgagct 1680gtggagacca ctgcccagag tgacaataag atcacatggt ccattatccg ggagcacatg 1740ggggagattc tctataaact ctcctccatg aaattcaagg atccagtgaa agatggcgag 1800gcaaagatca aggccgacta tgcacagctt cttgaagata tgcagaatgc attccgtagc 1860cttgaagact agaactgtga tttcttccct cttccacggc aagctcatac gtgtatattt 1920tcctgaattt ctcatctcca acccttagct tccatattgt gcagctttga gactagtgcc 1980tatgtgtgtt cttgttcatt ttgctgtttc tttggtaggt cttataaaac aaacattcct 2040ttgttccagt gtttgaagga gctagctccc ttacctttat ctgaagtggt gaatgtagtg 2100catatgatat acattgtaag atacacattg taacatgatc catactgtaa acttgtatgt 2160aaggtgacta ccccttccct catctccagt aaactgtaaa caggactact gcatgtactc 2220tgttgggaat ggaaggccag aactccatac cgtggatgga tgggtactta ggaaacaact 2280cagcatttgt agtcagacca cttgtaactt agtggtttgt tgagtaacca ttttgcagga 2340aatacttcca tttaaaaaca taaaagatta atgttccaat tatttgtatc aatcaggacc 2400atttttgtgg ggcacttggg aactatttgt ttttcaaaca gacatttgca agactgaaca 2460taatagataa atcagttacc tctgaaaatg tggaaagaaa gggaaaaaaa gaaccaggtg 2520gtcaaactta aattgacatc atcttgttaa agcatatttt atttcactaa gagaaattta 2580atatcagaga cttttatata ctcaattact aggaaacctt tttttaagta caatttaaaa 2640atcattgaaa atgtgatcca catcatagcc attcttttcc tcagacttag tcagacaagc 2700ttctcagagt ggtgggatgg ggattagaat accacagaca ctctgcagtg cctgcaggca 2760gtcggcccca ggacaaccac tgctgtagga gtttggggcc agggtggcat ggttttcaca 2820aggtacatgt gtcacgtgtt tgtttgcctg ttgacattct gaaaacagca agtttaccaa 2880ttgcagaaaa tactttctgt tttctctttc acatgctcag aaagcttctc aaaggtatct 2940ggtcacagca gcttccttcc tgttatagag gtaaaaggtg ttcttatatt taactggtaa 3000caaaaga 3007 13 823 PRT Rattus norvegicus Protein hypoxia-induciblefactor-1 alpha 13 Met Glu Gly Ala Gly Gly Glu Asn Glu Lys Lys Asn ArgMet Ser Ser 1 5 10 15 Glu Arg Arg Lys Glu Lys Ser Arg Asp Ala Ala ArgSer Arg Arg Ser 20 25 30 Lys Glu Ser Glu Val Phe Tyr Glu Leu Ala His GlnLeu Pro Leu Pro 35 40 45 His Asn Val Ser Ser His Leu Asp Lys Ala Ser ValMet Arg Leu Thr 50 55 60 Ile Ser Tyr Leu Arg Val Arg Lys Leu Leu Gly AlaGly Asp Leu Asp 65 70 75 80 Ile Glu Asp Glu Met Lys Ala Gln Met Asn CysPhe Tyr Leu Lys Ala 85 90 95 Leu Asp Gly Phe Val Met Val Leu Thr Asp AspGly Asp Met Ile Tyr 100 105 110 Ile Ser Asp Asn Val Asn Lys Tyr Met GlyLeu Thr Gln Phe Glu Leu 115 120 125 Thr Gly His Ser Val Phe Asp Phe ThrHis Pro Cys Asp His Glu Glu 130 135 140 Met Arg Glu Met Leu Thr His ArgAsn Gly Pro Val Arg Lys Gly Lys 145 150 155 160 Glu Gln Asn Thr Gln ArgSer Phe Phe Leu Arg Met Lys Cys Thr Leu 165 170 175 Thr Ser Arg Gly ArgThr Met Asn Ile Lys Ser Ala Thr Trp Lys Val 180 185 190 Leu His Cys ThrGly His Ile His Val Tyr Asp Thr Ser Ser Asn Gln 195 200 205 Pro Gln CysGly Tyr Lys Lys Pro Pro Met Thr Cys Leu Val Leu Ile 210 215 220 Cys GluPro Ile Pro His Pro Ser Asn Ile Glu Ile Pro Leu Asp Ser 225 230 235 240Lys Thr Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe Ser Tyr Cys 245 250255 Asp Glu Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu Glu Leu Leu 260265 270 Gly Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser Asp His Leu275 280 285 Thr Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val Thr ThrGly 290 295 300 Gln Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val Trp ValGlu Thr 305 310 315 320 Gln Ala Thr Val Ile Tyr Asn Thr Lys Asn Ser GlnPro Gln Cys Ile 325 330 335 Val Cys Val Asn Tyr Val Val Ser Gly Ile IleGln His Asp Leu Ile 340 345 350 Phe Ser Leu Gln Gln Thr Glu Ser Val LeuLys Pro Val Glu Ser Ser 355 360 365 Asp Met Lys Met Thr Gln Leu Phe ThrLys Val Glu Ser Glu Asp Thr 370 375 380 Ser Cys Leu Phe Asp Lys Leu LysLys Glu Pro Asp Ala Leu Thr Leu 385 390 395 400 Leu Ala Pro Ala Ala GlyAsp Thr Ile Ile Ser Leu Asp Phe Gly Ser 405 410 415 Asp Asp Thr Glu ThrGlu Asp Gln Gln Leu Glu Asp Val Pro Leu Tyr 420 425 430 Asn Asp Val MetPhe Pro Ser Ser Asn Glu Lys Leu Asn Ile Asn Leu 435 440 445 Ala Met SerPro Leu Pro Ala Ser Glu Thr Pro Lys Pro Leu Arg Ser 450 455 460 Ser AlaAsp Pro Ala Leu Asn Gln Glu Val Ala Leu Lys Leu Glu Ser 465 470 475 480Ser Pro Glu Ser Leu Gly Leu Ser Phe Thr Met Pro Gln Ile Gln Asp 485 490495 Gln Pro Ala Ser Pro Ser Asp Gly Ser Thr Arg Gln Ser Ser Pro Glu 500505 510 Pro Asn Ser Pro Ser Glu Tyr Cys Phe Asp Val Asp Ser Asp Met Val515 520 525 Asn Val Phe Lys Leu Glu Leu Val Glu Lys Leu Phe Ala Glu AspThr 530 535 540 Glu Ala Lys Asn Pro Phe Ser Ala Gln Asp Thr Asp Leu AspLeu Glu 545 550 555 560 Met Leu Ala Pro Tyr Ile Pro Met Asp Asp Asp PheGln Leu Arg Ser 565 570 575 Phe Asp Gln Leu Ser Pro Leu Glu Ser Asn SerPro Ser Pro Pro Ser 580 585 590 Val Ser Thr Val Thr Gly Phe Gln Gln ThrGln Leu Gln Lys Pro Thr 595 600 605 Ile Thr Val Thr Ala Thr Ala Thr AlaThr Thr Asp Glu Ser Lys Ala 610 615 620 Val Thr Lys Asp Asn Ile Glu AspIle Lys Ile Leu Ile Ala Ser Pro 625 630 635 640 Pro Ser Thr Gln Val ProGln Glu Met Thr Thr Ala Lys Ala Ser Ala 645 650 655 Tyr Ser Gly Thr HisSer Arg Thr Ala Ser Pro Asp Arg Ala Gly Lys 660 665 670 Arg Val Ile GluLys Thr Asp Lys Ala His Pro Arg Ser Leu Asn Leu 675 680 685 Ser Val ThrLeu Asn Gln Arg Asn Thr Val Pro Glu Glu Glu Leu Asn 690 695 700 Pro LysThr Ile Ala Leu Gln Asn Ala Gln Arg Lys Arg Lys Met Glu 705 710 715 720His Asp Gly Ser Leu Phe Gln Ala Ala Gly Ile Gly Thr Leu Leu Gln 725 730735 Gln Pro Gly Asp Arg Ala Pro Thr Met Ser Leu Ser Trp Lys Arg Val 740745 750 Lys Gly Tyr Ile Ser Ser Glu Gln Asp Gly Met Glu Gln Lys Thr Ile755 760 765 Phe Leu Ile Pro Ser Asp Leu Ala Cys Arg Leu Leu Gly Gln SerMet 770 775 780 Asp Glu Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys GluVal Asn 785 790 795 800 Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln GlyGlu Glu Leu Leu 805 810 815 Arg Ala Leu Asp Gln Val Asn 820 14 3718 DNARattus norvegicus cDNA hypoxia-inducible factor-1 alpha 14 gacaccgcgggcaccgattc gccatggagg gcgccggcgg cgagaacgag aagaaaaata 60 ggatgagttccgaacgtcga aaagaaaagt ctagggatgc agcacgatct cggcgaagca 120 aagagtctgaagttttttat gagcttgctc atcagttgcc acttccccac aacgtgagct 180 cccatcttgataaagcttct gttatgaggc tcaccatcag ttacttacgt gtgaggaaac 240 ttctaggtgctggtgatctt gacattgaag atgaaatgaa agcacagatg aactgctttt 300 atctgaaagccctggatggc tttgttatgg tgctaacaga tgatggtgac atgatttaca 360 tttctgataacgtgaacaaa tacatggggt tgactcagtt tgaactaact ggacacagtg 420 tgtttgattttacccatcca tgtgaccatg aggaaatgag agaaatgctt acacacagaa 480 atggcccagtgagaaagggg aaagaacaaa acacgcagcg aagctttttt ctcagaatga 540 aatgtaccctaacaagccgg gggaggacga tgaacatcaa gtcagcaacg tggaaggtgc 600 tgcactgcacaggccacatt catgtgtatg ataccagcag taaccagccg cagtgtggct 660 acaagaaaccgcctatgacg tgcttggtgc tgatttgtga acccattcct catccatcaa 720 acattgaaattcctttagac agcaagacat ttctcagtcg acacagcctc gatatgaaat 780 tttcttactgtgatgaaagg attactgagt tgatgggtta tgagccagaa gaacttttgg 840 gccgttcaatttatgaatat tatcatgctt tggactctga tcatctgacc aaaactcatc 900 atgacatgtttactaaagga caagtcacca caggacagta caggatgctt gcaaaaagag 960 gtggatatgtctgggttgag actcaagcaa ctgttatata taatacgaag aactctcagc 1020 cacagtgcattgtgtgtgtg aattatgttg taagtggtat tattcagcac gacttgattt 1080 tctcccttcaacaaacagaa tctgtcctca aaccagttga atcttcagat atgaaaatga 1140 cccagctgttcactaaagtg gaatctgagg acacgagctg cctcttcgac aagcttaaga 1200 aagagcccgatgccctgact ctgctagctc cagcggctgg ggacacgatc atatcactgg 1260 acttcggcagcgatgacacg gaaactgaag accaacaact tgaagatgtc ccgttgtaca 1320 atgatgtaatgttcccctct tctaatgaga aattaaatat aaatctggca atgtctccat 1380 tacctgcctctgaaactcca aagccacttc gaagtagtgc tgatcctgca ctgaatcaag 1440 aggttgcattgaagttagag tcaagcccag agtcactggg actttctttt accatgcccc 1500 agattcaagatcagccagca agtccttctg atggaagcac tagacaaagc tcacctgagc 1560 ctaacagtcccagtgagtac tgctttgatg tggacagcga tatggtcaat gtattcaagt 1620 tggaactggtggaaaaactg tttgctgaag acacagaagc gaagaatcca ttttcagctc 1680 aggacactgatttagacttg gaaatgctgg ctccctatat cccaatggat gatgatttcc 1740 agttacgttcctttgatcag ttgtcaccat tagagagcaa ttctccaagc cctccgagtg 1800 tgagcacagttacaggattc cagcagaccc agttacagaa acctaccatc actgtcactg 1860 ccaccgcaactgccaccact gatgaatcaa aagcagtgac gaaggacaat atagaagaca 1920 ttaaaatactgattgcatct ccaccttcta cccaagtacc tcaagaaatg accactgcta 1980 aggcatcagcatacagtggt actcacagtc ggacagcctc accagacaga gcaggaaaga 2040 gagtcatagaaaaaacagac aaagctcatc caaggagcct taacctatct gtcactttga 2100 atcaaagaaatactgttcct gaagaagaat taaacccaaa gacaatagct ttgcagaatg 2160 ctcagaggaagcgaaaaatg gaacatgatg gctccctttt tcaagcagca ggaattggaa 2220 cgttactgcagcaaccaggt gaccgtgccc ctactatgtc gctttcttgg aaacgagtga 2280 aaggatacatatctagtgaa caggatggaa tggagcagaa gacaattttt ttaataccct 2340 ctgatttagcatgtagactg ctggggcagt caatggatga gagtggatta ccacagctga 2400 ccagttacgattgtgaagtt aatgctccca tacaaggcag cagaaaccta ctgcagggtg 2460 aagaattactcagagctttg gatcaagtta actgagcttt tcctaatctc attcctttga 2520 ttgttaatttttgtgttcag ttgttgttgt tgtctgtggg gtttcgtttc tgttggttgt 2580 tttggacactggtggctcag cagtctattt atattttcta tatctcattt agaggcctgg 2640 ctacagtactgcaccaactc agatagttta gtttgggccc cttcctcctt cattttcact 2700 gatgctctttttaccatgtc cttcgaatgc cagatcacag cacattcaca gctccccagc 2760 atttcaccaatgcattgctg tagtgtcgtt taaaatgcac ctttttattt atttattttt 2820 ggtgagggagtttgtccctt attgaattat ttttaatgaa atgccaatat aattttttaa 2880 gaaggcagtaaatcttcatc atgatgatag gcagttgaaa attttttact catttttttc 2940 atgttttacatgaaaataat gctttgccag cagtacatgg tagccacaat tgcacaatat 3000 attttcttaaaaataccagc agttactcat gcatatattc tgcatttata aaactagttt 3060 ttaagaagaaactttttttg gcctatggaa ttgttaagcc tggatcatga tgctgttgat 3120 cttataatgattcttaaact gtatggtttc tttatatggg taaagccatt tacatgatat 3180 agagagatatgcttatatct ggaaggtata tggcatttat ttggataaaa ttctcaattg 3240 agaagttatctggtgtttct ttactttacc ggctcaaaag aaaacagtcc ctatgtagtt 3300 gtggaagcttatgctaatat tgtgtaattg atattaaaca ttaaatgttc tgcctatcct 3360 gttggtataaagacattttg agcatactgt aaacaaaaaa atcatgcatt gttagtaaaa 3420 ttgcctagtatgttaatttg ttgaaaatac gatgtttggt tttatgcact ttgtcgctat 3480 taacatcctttttttcatat agatttcaat aattgagtaa ttttagaagc attattttag 3540 aaatatagagttgtcatagt aaacatcttg tttttttttc tttttttcta tgtacattgt 3600 ataaatttttcattcccttg ctctttgtag ttgggtctaa cactaactgt actgttttgt 3660 tatatcaaataaacatcttc tgtggaccag gaaaaaaaaa aaaaaaaaaa aaaaaaaa 3718 15 227 PRTRattus norvegicus Protein cytochrome c oxidase subunit II 15 Met Ala TyrPro Phe Gln Leu Gly Leu Gln Asp Ala Thr Ser Pro Ile 1 5 10 15 Met GluGlu Leu Thr Asn Phe His Asp His Thr Leu Met Ile Val Phe 20 25 30 Leu IleSer Ser Leu Val Leu Tyr Ile Ile Ser Leu Met Leu Thr Thr 35 40 45 Lys LeuThr His Thr Ser Thr Met Asp Ala His Glu Val Glu Thr Ile 50 55 60 Trp ThrIle Leu Pro Ala Val Ile Leu Ile Leu Ile Ala Leu Pro Ser 65 70 75 80 LeuArg Ile Leu Tyr Met Met Asp Glu Ile Asn Asn Pro Val Leu Thr 85 90 95 ValLys Thr Met Gly His Gln Trp Tyr Trp Ser Tyr Glu Tyr Thr Asp 100 105 110Tyr Glu Asp Leu Cys Phe Asp Ser Tyr Met Ile Pro Thr Asn Asp Leu 115 120125 Lys Leu Gly Glu Leu Arg Leu Leu Glu Val Asp Asn Arg Val Val Leu 130135 140 Pro Met Glu Leu Pro Ile Arg Met Leu Ile Ser Ser Glu Asp Val Leu145 150 155 160 His Ser Trp Pro Ile Pro Ser Leu Gly Leu Lys Thr Asp AlaIle Pro 165 170 175 Gly Arg Pro Asn Gln Ala Thr Val Thr Ser Asn Arg LeuGly Leu Phe 180 185 190 Tyr Gly Gln Cys Ser Glu Ile Cys Gly Ser Asn HisSer Phe Met Leu 195 200 205 Ile Val Leu Glu Met Val Pro Leu Lys Tyr PheGlu Asn Trp Ser Ala 210 215 220 Ser Met Ile 225 16 686 DNA Rattusnorvegicus cDNA cytochrome c oxidase subunit II 16 acatggctta cccatttcaacttggcttac aagacgccac atcaccaatc atagaagaac 60 ttacaaactt tcatgaccacaccctaataa ttgtattcct catcagctcc ctagtacttt 120 atattatttc actaatactaacaacaaaac taacacacac aagcacaata gacgcccatg 180 aagtagaaac aatttgaacaattctcccag ctgtcattct tattctaatc gcccttccct 240 ccctacgaat tctatacataatagacgaga ttaataaccc agttctaaca gtaaaaacta 300 taggacacca atgatactgaagctatgaat atactgacta tgaagaccta tgctttgact 360 cctacataat cccaaccaatgacctaaaac taggtgaact tcgcttatta gaagttgata 420 atcgggtagt cttaccaatagaacttccaa ttcgtatact aatctcatcc gaagacgtcc 480 tgcactcatg acccatcccttcactagggt taaaaaccga cgcaatcccc ggccgcccga 540 accaagctac agtcacatcaaaccgactag gtctattcta tggccaatgc tctgaaattt 600 gcggctcaaa tcacagcttcatactcattg tactagaaat agtgcctcta aaatatttcg 660 aaaactgatc agcttctataatttaa 686 17 260 PRT Rattus norvegicus Protein HYDROXYACYLGLUTATHIONEHYDROLASE 17 Met Lys Ile Glu Leu Leu Pro Ala Leu Thr Asp Asn Tyr Met TyrLeu 1 5 10 15 Ile Ile Asp Glu Asp Thr Gln Glu Ala Ala Val Val Asp ProVal Gln 20 25 30 Pro Gln Lys Val Ile Glu Thr Val Lys Lys His Arg Val LysLeu Thr 35 40 45 Thr Val Leu Thr Thr His His His Trp Asp His Ala Gly GlyAsn Glu 50 55 60 Lys Leu Val Lys Leu Glu Pro Gly Leu Lys Val Tyr Gly GlyAsp Asp 65 70 75 80 Arg Ile Gly Ala Leu Thr His Lys Val Thr His Leu SerThr Leu Glu 85 90 95 Val Gly Ser Leu Ser Val Lys Cys Leu Ser Thr Pro CysHis Thr Ser 100 105 110 Gly His Ile Cys Tyr Phe Val Ser Lys Pro Gly SerSer Glu Pro Ser 115 120 125 Ala Val Phe Thr Gly Asp Thr Leu Phe Val AlaGly Cys Gly Lys Phe 130 135 140 Tyr Glu Gly Thr Ala Asp Glu Met Tyr LysAla Leu Leu Glu Val Leu 145 150 155 160 Gly Arg Leu Pro Pro Asp Thr LysVal Ile Cys Gly His Glu Tyr Thr 165 170 175 Val Asn Asn Leu Lys Phe AlaArg His Val Glu Pro Gly Asn Thr Ala 180 185 190 Val Gln Glu Lys Leu AlaTrp Ala Lys Glu Lys Asn Ala Ile Gly Glu 195 200 205 Pro Thr Val Pro SerThr Leu Ala Glu Glu Phe Thr Tyr Asn Pro Phe 210 215 220 Met Arg Val LysGlu Lys Thr Val Gln Gln His Ala Gly Glu Thr Asp 225 230 235 240 Pro ValThr Thr Met Arg Ala Ile Arg Arg Glu Lys Asp Gln Phe Lys 245 250 255 ValPro Arg Asp 260 18 504 PRT Homo sapiens Protein 26S PROTEASOME SUBUNITS5B 18 Met Ala Ala Gln Ala Leu Ala Leu Leu Arg Glu Val Ala Arg Leu Glu 15 10 15 Ala Pro Leu Glu Glu Leu Arg Ala Leu His Ser Val Leu Gln Ala Val20 25 30 Pro Leu Asn Glu Leu Arg Gln Gln Ala Ala Glu Leu Arg Leu Gly Pro35 40 45 Leu Phe Ser Leu Leu Asn Glu Asn His Arg Glu Lys Thr Thr Leu Cys50 55 60 Val Ser Ile Leu Glu Arg Leu Leu Gln Ala Met Glu Pro Val His Val65 70 75 80 Ala Arg Asn Leu Arg Val Asp Leu Gln Arg Gly Leu Ile His ProAsp 85 90 95 Asp Ser Val Lys Ile Leu Thr Leu Ser Gln Ile Gly Arg Ile ValGlu 100 105 110 Asn Ser Asp Ala Val Thr Glu Ile Leu Asn Asn Ala Glu LeuLeu Lys 115 120 125 Gln Ile Val Tyr Cys Ile Gly Gly Glu Asn Leu Ser ValAla Lys Ala 130 135 140 Ala Ile Lys Ser Leu Ser Arg Ile Ser Leu Thr GlnAla Gly Leu Glu 145 150 155 160 Ala Leu Phe Glu Ser Asn Leu Leu Asp AspLeu Lys Ser Val Met Lys 165 170 175 Thr Asn Asp Ile Val Arg Tyr Arg ValTyr Glu Leu Ile Ile Glu Ile 180 185 190 Ser Ser Val Ser Pro Glu Ser LeuAsn Tyr Cys Thr Thr Ser Gly Leu 195 200 205 Val Thr Gln Leu Leu Arg GluLeu Thr Gly Glu Asp Val Leu Val Arg 210 215 220 Ala Thr Cys Ile Glu MetVal Thr Ser Leu Ala Tyr Thr His His Gly 225 230 235 240 Arg Gln Tyr LeuAla Gln Glu Gly Val Ile Asp Gln Ile Ser Asn Ile 245 250 255 Ile Val GlyAla Asp Ser Asp Pro Phe Ser Ser Phe Tyr Leu Pro Gly 260 265 270 Phe ValLys Phe Phe Gly Asn Leu Ala Val Met Asp Ser Pro Gln Gln 275 280 285 IleCys Glu Arg Tyr Pro Ile Phe Val Glu Lys Val Phe Glu Met Ile 290 295 300Glu Ser Gln Asp Pro Thr Met Ile Gly Val Ala Val Asp Thr Val Gly 305 310315 320 Ile Leu Gly Ser Asn Val Glu Gly Lys Gln Val Leu Gln Lys Thr Gly325 330 335 Thr Arg Phe Glu Arg Leu Leu Met Arg Ile Gly His Gln Ser LysAsn 340 345 350 Ala Pro Val Glu Leu Lys Ile Arg Cys Leu Asp Ala Ile SerSer Leu 355 360 365 Leu Tyr Leu Pro Pro Glu Gln Gln Thr Asp Asp Leu LeuArg Met Thr 370 375 380 Glu Ser Trp Phe Ser Ser Leu Ser Arg Asp Pro LeuGlu Leu Phe Arg 385 390 395 400 Gly Ile Ser Ser Gln Pro Phe Pro Glu LeuHis Cys Ala Ala Leu Lys 405 410 415 Val Phe Thr Ala Ile Ala Asn Gln ProTrp Ala Gln Lys Leu Met Phe 420 425 430 Asn Ser Pro Gly Phe Val Glu TyrVal Val Asp Arg Ser Val Glu His 435 440 445 Asp Lys Ala Ser Lys Asp AlaLys Tyr Glu Leu Val Lys Ala Leu Ala 450 455 460 Asn Ser Lys Thr Ile AlaGlu Ile Phe Gly Asn Pro Asn Tyr Leu Arg 465 470 475 480 Leu Arg Thr TyrLeu Ser Glu Gly Pro Tyr Tyr Val Lys Pro Val Ser 485 490 495 Thr Thr AlaVal Glu Gly Ala Glu 500 19 1128 PRT Homo sapiens Protein Nck-associatedprotein 1 (Nap 1) 19 Met Ser Arg Ser Val Leu Gln Pro Ser Gln Gln Lys LeuAla Glu Lys 1 5 10 15 Leu Thr Ile Leu Asn Asp Arg Gly Val Gly Met LeuThr Arg Leu Tyr 20 25 30 Asn Ile Lys Lys Ala Cys Gly Asp Pro Lys Ala LysPro Ser Tyr Leu 35 40 45 Ile Asp Lys Asn Leu Glu Ser Ala Val Lys Phe IleVal Arg Lys Phe 50 55 60 Pro Ala Val Glu Thr Arg Asn Asn Asn Gln Gln LeuAla Gln Leu Gln 65 70 75 80 Lys Glu Lys Ser Glu Ile Leu Lys Asn Leu AlaLeu Tyr Tyr Phe Thr 85 90 95 Phe Val Asp Val Met Glu Phe Lys Asp His ValCys Glu Leu Leu Asn 100 105 110 Thr Ile Asp Val Cys Gln Val Phe Phe AspIle Thr Val Asn Phe Asp 115 120 125 Leu Thr Lys Asn Tyr Leu Asp Leu IleIle Thr Tyr Thr Thr Leu Met 130 135 140 Ile Leu Leu Ser Arg Ile Glu GluArg Lys Ala Ile Ile Gly Leu Tyr 145 150 155 160 Asn Tyr Ala His Glu MetThr His Gly Ala Ser Asp Arg Glu Tyr Pro 165 170 175 Arg Leu Gly Gln MetIle Val Asp Tyr Glu Asn Pro Leu Lys Lys Met 180 185 190 Met Glu Glu PheVal Pro His Ser Lys Ser Leu Ser Asp Ala Leu Ile 195 200 205 Ser Leu GlnMet Val Tyr Pro Arg Arg Asn Leu Ser Ala Asp Gln Trp 210 215 220 Arg AsnAla Gln Leu Leu Ser Leu Ile Ser Ala Pro Ser Thr Met Leu 225 230 235 240Asn Pro Ala Gln Ser Asp Thr Met Pro Cys Glu Tyr Leu Ser Leu Asp 245 250255 Ala Met Glu Lys Trp Ile Ile Phe Gly Phe Ile Leu Cys His Gly Ile 260265 270 Leu Asn Thr Asp Ala Thr Ala Leu Asn Leu Trp Lys Leu Ala Leu Gln275 280 285 Ser Ser Ser Cys Leu Ser Leu Phe Arg Asp Glu Val Phe His IleHis 290 295 300 Lys Ala Ala Glu Asp Leu Phe Val Asn Ile Arg Gly Tyr AsnLys Arg 305 310 315 320 Ile Asn Asp Ile Arg Glu Cys Lys Glu Ala Ala ValSer His Ala Gly 325 330 335 Ser Met His Arg Glu Arg Arg Lys Phe Leu ArgSer Ala Leu Lys Glu 340 345 350 Leu Ala Thr Val Leu Ser Asp Gln Pro GlyLeu Leu Gly Pro Lys Ala 355 360 365 Leu Phe Val Phe Met Ala Leu Ser PheAla Arg Asp Glu Ile Ile Trp 370 375 380 Leu Leu Arg His Ala Asp Asn MetPro Lys Lys Ser Ala Asp Asp Phe 385 390 395 400 Ile Asp Lys His Ile AlaGlu Leu Ile Phe Tyr Met Glu Glu Leu Arg 405 410 415 Ala His Val Arg LysTyr Gly Pro Val Met Gln Arg Tyr Tyr Val Gln 420 425 430 Tyr Leu Ser GlyPhe Asp Ala Val Val Leu Asn Glu Leu Val Gln Asn 435 440 445 Leu Ser ValCys Pro Glu Asp Glu Ser Ile Ile Met Ser Ser Phe Val 450 455 460 Asn ThrMet Thr Ser Leu Ser Val Lys Gln Val Glu Asp Gly Glu Val 465 470 475 480Phe Asp Phe Arg Gly Met Arg Leu Asp Trp Phe Arg Leu Gln Ala Tyr 485 490495 Thr Ser Val Ser Lys Ala Ser Leu Gly Leu Ala Asp His Arg Glu Leu 500505 510 Gly Lys Met Met Asn Thr Ile Ile Phe His Thr Lys Met Val Asp Ser515 520 525 Leu Val Glu Met Leu Val Glu Thr Ser Asp Leu Ser Ile Phe CysPhe 530 535 540 Tyr Ser Arg Ala Phe Glu Lys Met Phe Gln Gln Cys Leu GluLeu Pro 545 550 555 560 Ser Gln Ser Arg Tyr Ser Ile Ala Phe Pro Leu LeuCys Thr His Phe 565 570 575 Met Ser Cys Thr His Glu Leu Cys Pro Glu GluArg His His Ile Gly 580 585 590 Asp Arg Ser Leu Ser Leu Cys Asn Met PheLeu Asp Glu Met Ala Lys 595 600 605 Gln Ala Arg Asn Leu Ile Thr Asp IleCys Thr Glu Gln Cys Thr Leu 610 615 620 Ser Asp Gln Leu Leu Pro Lys HisCys Ala Lys Thr Ile Ser Gln Ala 625 630 635 640 Val Asn Lys Lys Ser LysLys Gln Thr Gly Lys Lys Gly Glu Pro Glu 645 650 655 Arg Glu Lys Pro GlyVal Glu Ser Met Arg Lys Asn Arg Leu Val Val 660 665 670 Thr Asn Leu AspLys Leu His Thr Ala Leu Ser Glu Leu Cys Phe Ser 675 680 685 Ile Asn TyrVal Pro Asn Met Val Val Trp Glu His Thr Phe Thr Pro 690 695 700 Arg GluTyr Leu Thr Ser His Leu Glu Ile Arg Phe Thr Lys Ser Ile 705 710 715 720Val Gly Met Thr Met Tyr Asn Gln Ala Thr Gln Glu Ile Ala Lys Pro 725 730735 Ser Glu Leu Leu Thr Ser Val Arg Ala Tyr Met Thr Val Leu Gln Ser 740745 750 Ile Glu Asn Tyr Val Gln Ile Asp Ile Thr Arg Val Phe Asn Asn Val755 760 765 Leu Leu Gln Gln Thr Gln His Leu Asp Ser His Gly Glu Pro ThrIle 770 775 780 Thr Ser Leu Tyr Thr Asn Trp Tyr Leu Glu Thr Leu Leu ArgGln Val 785 790 795 800 Ser Asn Gly His Ile Ala Tyr Phe Pro Ala Met LysAla Phe Val Asn 805 810 815 Leu Pro Thr Glu Asn Glu Leu Thr Phe Asn AlaGlu Glu Tyr Ser Asp 820 825 830 Ile Ser Glu Met Arg Ser Leu Ser Glu LeuLeu Gly Pro Tyr Gly Met 835 840 845 Lys Phe Leu Ser Glu Ser Leu Met TrpHis Ile Ser Ser Gln Val Ala 850 855 860 Glu Leu Lys Lys Leu Val Val GluAsn Val Asp Val Leu Thr Gln Met 865 870 875 880 Arg Thr Ser Phe Asp LysPro Asp Gln Met Ala Ala Leu Phe Lys Arg 885 890 895 Leu Ser Ser Val AspSer Val Leu Lys Arg Met Thr Ile Ile Gly Val 900 905 910 Ile Leu Ser PheArg Ser Leu Ala Gln Glu Ala Leu Arg Asp Val Leu 915 920 925 Ser Tyr HisIle Pro Phe Leu Val Ser Ser Ile Glu Asp Phe Lys Asp 930 935 940 His IlePro Arg Glu Thr Asp Met Lys Val Ala Met Asn Val Tyr Glu 945 950 955 960Leu Ser Ser Ala Ala Gly Leu Pro Cys Glu Ile Asp Pro Ala Leu Val 965 970975 Val Ala Leu Ser Ser Gln Lys Ser Glu Asn Ile Ser Pro Glu Glu Glu 980985 990 Tyr Lys Ile Ala Cys Leu Leu Met Val Phe Val Ala Val Ser Leu Pro995 1000 1005 Thr Leu Ala Ser Asn Val Met Ser Gln Tyr Ser Pro Ala IleGlu 1010 1015 1020 Gly His Cys Asn Asn Ile His Cys Leu Ala Lys Ala IleAsn Gln 1025 1030 1035 Ile Ala Ala Ala Leu Phe Thr Ile His Lys Gly SerIle Glu Asp 1040 1045 1050 Arg Leu Lys Glu Phe Leu Ala Leu Ala Ser SerSer Leu Leu Lys 1055 1060 1065 Ile Gly Gln Glu Thr Asp Lys Thr Thr ThrArg Asn Arg Glu Ser 1070 1075 1080 Val Tyr Leu Leu Leu Asp Met Ile ValGln Glu Ser Pro Phe Leu 1085 1090 1095 Thr Met Asp Leu Leu Glu Ser CysPhe Pro Tyr Val Leu Leu Arg 1100 1105 1110 Asn Ala Tyr His Ala Val TyrLys Gln Ser Val Thr Ser Ser Ala 1115 1120 1125 20 4218 DNA Homo sapienscDNA Nck-associated protein 1 (Nap 1) 20 cggcggcacc agcaccaccatgtcgcgctc agtgctgcag cccagtcagc agaagctggc 60 ggagaagctc accatcctcaacgaccgggg cgtcggcatg ctcacccgcc tctacaacat 120 caagaaggca tgtggagaccccaaggcaaa accatcctat cttatcgaca aaaacctgga 180 atctgctgtg aaattcatagtcagaaaatt ccctgctgta gaaacccgca acaacaatca 240 acagcttgca caactacagaaagaaaaatc agagattctg aaaaatctgg cattatatta 300 cttcacattt gtagatgttatggaatttaa ggaccatgtt tgtgaattgc tgaatactat 360 tgacgtttgc caagtcttctttgatattac tgtaaacttt gatttaacaa agaactactt 420 agatttaatt ataacctatacaacactaat gatactgctg tctcgaattg aagaaaggaa 480 ggcaatcatt ggattatacaactatgccca tgaaatgact catggagcaa gtgacagaga 540 atacccacgc cttggccagatgattgtgga ttatgaaaac cctttaaaga agatgatgga 600 agaatttgta ccccatagcaagtctctttc agatgcacta atttctcttc aaatggtata 660 tcctcgaagg aatctttcagctgaccagtg gagaaatgcc cagttattga gcctcatcag 720 tgcacctagt acaatgcttaatccagcaca gtccgacact atgccttgtg aatacctctc 780 tttggatgca atggaaaagtggattatctt tggctttatt ttgtgccatg ggatcctaaa 840 tactgacgct acagcactgaacctttggaa actagctctt caaagtagct cttgcctctc 900 tctctttcgg gatgaagttttccacattca caaagctgca gaagacttat ttgtaaacat 960 acgaggctat aataaacgtattaatgacat aagagaatgc aaggaggcag ccgtgtcaca 1020 tgctggttca atgcacagagaaagacgcaa gtttttaaga tctgcactga aggaattggc 1080 tactgtcctc tctgatcaacctggattgct aggtcccaag gcactttttg tttttatggc 1140 attatccttt gcccgtgatgaaatcatctg gctacttcgt catgcagata acatgccaaa 1200 gaagagtgca gacgactttatagataagca cattgctgaa ttaatatttt acatggaaga 1260 acttagagca catgtgaggaaatacggacc tgtaatgcag aggtattacg tgcagtacct 1320 ttctggcttt gatgctgttgtcctcaatga actcgtgcag aatctttctg tttgccctga 1380 agatgaatca atcatcatgtcctcttttgt taacactatg acttccctaa gtgtaaaaca 1440 agttgaagat ggggaagtatttgatttcag aggaatgaga ttagattggt ttaggttaca 1500 ggcatatact agtgtctcaaaggcttcact tggccttgca gatcacagag aacttgggaa 1560 gatgatgaat acaataatttttcatacaaa aatggtagat tccttggtgg aaatgttggt 1620 ggaaacatca gatctctccatattttgttt ttatagtcgt gcttttgaga agatgtttca 1680 acagtgtttg gagttaccctctcaatcaag atactcaatt gcatttccac tactttgcac 1740 tcattttatg agttgcacgcatgaactatg tccagaagag cgacatcata ttggagatcg 1800 cagtctttcc ttatgtaatatgttcctaga tgaaatggcc aaacaagctc gaaatctcat 1860 cactgatatt tgcacagaacagtgtaccct tagtgaccag ttgctaccca agcattgtgc 1920 caaaactatc agtcaagcagtgaataagaa atcaaaaaag cagactggta agaaagggga 1980 acctgaaagg gagaaaccaggtgttgagag catgaggaaa aacaggctgg ttgtgaccaa 2040 ccttgataaa ttgcacactgcactttctga gttatgcttc tctataaatt atgtaccaaa 2100 catggtggta tgggaacatacctttacccc acgagaatat ttgacttctc atctggaaat 2160 acgctttacc aagtcaattgttgggatgac tatgtataat caagccacac aggaaattgc 2220 aaaaccttca gaacttctaacaagtgtaag agcatacatg accgtactcc agtcaataga 2280 aaactatgtg cagattgatattacaagagt atttaataat gtgcttcttc aacaaacaca 2340 acatttagac agtcatggagagccaaccat tacaagtcta tacacaaatt ggtatttgga 2400 aactttgtta cgacaagtcagcaatggcca tatagcatat tttcctgcaa tgaaagcgtt 2460 tgtgaactta cctacagaaaatgaattaac attcaatgca gaggaatatt ctgacatatc 2520 agaaatgagg tcattatcagaactactagg cccatatggt atgaagtttc taagtgaaag 2580 ccttatgtgg catatttcatcacaagttgc tgaacttaag aaacttgtgg tggagaatgt 2640 tgatgtgtta acacaaatgaggaccagctt tgacaaacca gaccagatgg ctgcactgtt 2700 taaaagatta tcatctgttgacagtgtctt gaagaggatg acaataattg gtgtaatttt 2760 atccttccga tcattggcacaagaagcact tagagatgtg ttatcctacc acattccttt 2820 tcttgtaagt tcaattgaagattttaagga tcacattcca agggaaactg atatgaaggt 2880 tgcaatgaat gtgtatgagttatcatcagc tgccggatta ccttgtgaga ttgatcctgc 2940 attggtcgta gctctttcttcacaaaaatc ggaaaacatt agtccagaag aagagtataa 3000 aattgcctgc cttctcatggtgtttgtggc agtttctttg ccaacactgg ccagtaatgt 3060 gatgtctcag tacagccctgctatagaagg gcattgcaac aacatacatt gcttggccaa 3120 agccatcaac cagattgctgcagctttgtt tacaattcac aaaggaagca ttgaagaccg 3180 tcttaaagaa tttctggcgcttgcatcctc cagtctactg aaaattggcc aggagacaga 3240 taaaactaca acaagaaatagagaatctgt ttatttactg ctagatatga ttgtacaaga 3300 atctccattc cttacaatggatcttttgga atcttgtttt ccttatgtct tgctgagaaa 3360 tgcataccat gctgtctacaaacaaagtgt tacatcttct gcataaaatt acctacttaa 3420 tcaagataag cacgcatttttgttgccttg gttttacctg tagactgtgg aactatttta 3480 ccttaagacc tgaaaaagttttgtggatta taaatttctt tcatacggtt gtattttctg 3540 atcattggtt tcttaatatggttgtactac agtatacttg gttgatttag gttgcacatt 3600 cactgaattc actgagattattcctataat tttaaagtat catttatttg aaaaacatac 3660 attatcaaca tgtttttgatatttgataat gaaaaaaatc tttgcttgtt tatttctgaa 3720 aaagaactgt atttagtgattattttagat agtgatatta tagcattcat ctgtgtgtaa 3780 attatttcat atagggaagagttctgatct gtacctatgg ttcttattga aaacaacatt 3840 ggatgtgcat ttctgtgatgttatgaatac atttctactt tattttgaaa catttgccaa 3900 actaaatact gtaacactgtataacattta aaaatgttaa agaactgctt agtattagaa 3960 gcagatcatt tcccaaaattctaagagcag cagcatatgt tgttgcttgt ataaagccta 4020 gcgataattt ttagactaacttccatggtg ccctgttggc attagcacta ccattgtacc 4080 ctgctgtata ataaacaatcttagacattt atcaactgtt gatacaaatg ttagtcccta 4140 accacttttt atatatgttttaaatttttg aaattcaagt gtaccttcca taacataaaa 4200 taaacactag actgtatc4218 21 166 PRT Mus musculus Protein Cofilin 21 Met Ala Ser Gly Val ThrVal Asn Asp Glu Val Ile Lys Val Phe Asn 1 5 10 15 Asp Met Lys Val ArgLys Ser Ser Thr Gln Glu Glu Ile Lys Lys Arg 20 25 30 Lys Lys Ala Val LeuPhe Cys Leu Ser Asp Asp Lys Arg Gln Ile Ile 35 40 45 Val Glu Glu Ala LysGln Ile Leu Val Gly Asp Ile Gly Asp Thr Val 50 55 60 Glu Asp Pro Tyr ThrSer Phe Val Lys Leu Leu Pro Leu Asn Asp Cys 65 70 75 80 Arg Tyr Ala LeuTyr Asp Ala Thr Tyr Glu Thr Lys Glu Ser Lys Lys 85 90 95 Glu Asp Leu ValPhe Ile Phe Trp Ala Pro Glu Ser Ala Pro Leu Lys 100 105 110 Ser Lys MetIle Tyr Ala Ser Ser Lys Asp Ala Ile Lys Lys Lys Phe 115 120 125 Thr GlyIle Lys His Glu Trp Gln Val Asn Gly Leu Asp Asp Ile Lys 130 135 140 AspArg Ser Thr Leu Gly Glu Lys Leu Gly Gly Ser Val Val Val Ser 145 150 155160 Leu Glu Gly Lys Pro Leu 165 22 2974 DNA Mus musculus cDNA Cofilin 22cgcccggctg cagctcccgg cgtgccctgc actctctgct gcccgccgcc gacccctcct 60tcttctcgtc ccagtgccac cgagccggag tccgagccac cgccgccgca gccacttcag 120ccgcgggcac tatggcatct ggagttacag tgaatgatga agtcatcaaa gtttttaatg 180atatgaaagt aagaaaatct tctacacagg aggagatcaa aaaaagaaag aaagcagttc 240tcttctgttt gagcgatgac aaaagacaaa taattgtaga ggaagccaag cagatcttgg 300tgggtgacat tggtgacact gtagaggacc cctacacatc ttttgtgaag ttgttgcctc 360tgaatgattg ccgatatgct ttgtacgatg ccacgtacga aacaaaagag tctaagaaag 420aagacctagt atttatattc tgggctcctg aaagtgcacc gttaaaaagc aagatgattt 480atgctagctc taaagatgcc attaaaaaga aatttacagg tattaaacat gagtggcaag 540taaatggctt ggacgatatt aaggaccgct cgacgctggg agagaaactg gggggcagtg 600ttgtagtttc ccttgaagga aagccactat aaaataatag ccaagtgcca tttgatctta 660aggggcttac acgtatctct ccagctcagt ccactggaat tgtattaggt tttgtttttt 720ttgtttattc ccttttcact ggtcccgttc gtgaatgagt gaatataaga agcctgtcag 780tattgccatg agactgtttc atatggttac ttttctgtat tcccaaggaa tgccttcctg 840tcttatttta gccaaaacaa actggttcca tgccttcctt gcagtgagcg ttacaatgga 900tgtggttgtc aatgtgaata gcttagagta ctacaaaggg taagctaact gaatgccttg 960aaaatattat ccactggtcg gtcatatggg agacttgttt cagtattatt tatagttgca 1020cttgattacc gttctctgag gcactggagc cttcatacac ctcacctgcc ttggcaagcc 1080tatttttgtg acctggcagc acagatttaa cactattcgt taaaagcact tttttttaat 1140gcgtttaatc ccttataaag aatgccaatt aagttttatt acctgtcatc aatttatcct 1200agtatctcag tgttcattct tcttgccttc atattttttt caaagaaaca gctgtgctaa 1260tgtctttggt ttcccgatga gtgtacacta ctgtataatt tatgtttacc atatgagtct 1320tgaaacacta cagatatttt gaatatcagt catgatggca atttctgtat aaaagagcct 1380taaatggaac attgttttga gatcaaactc cctaccctca caaaagtggc cacgttgcaa 1440taaaaattgt ggcagattac agaatgttgc cttgttttcc ttggaaattt tgcaaattgt 1500tatgtgaaat tttagggtaa cggtgattaa gctctgcact ggtatttgga attttttttc 1560ctttaatctt tggtttaaaa acatcttaaa atcacttata tacaatcatt aaaagagtgg 1620taattttata aatgcttatt tatgttataa aatggagatc agaaaaaaat tctttttgca 1680ctttggccta tccagtatct tatttattct ctagataagc taggatatta atccagagtt 1740acattactga gaattgagta gtataagtag gatgttttta ttacttggtc ataatgaaaa 1800taatttgtaa aatgtcattc gaaggttaat gatgattgtg atgtttagga atgtttgtct 1860cagccacagt tccttcatag cttttccaaa atgaattggg aaaaaaaatc gtatagcagt 1920cttaaagctt agtaatggaa cttggctgtg gcccagagct ttctccttat agagaatttg 1980atctgctccg tgtgtgctct ctgctattag ccggagctat ttatggcaaa cacatgcttt 2040tgtatcttgt catagtcatc cacagatggc aaaactggac ttgattctac tggcatgtaa 2100gacaggcgtg ctagtgagca gtcgtgtgtg gctctggact ctgaccccag agctctgaag 2160aatgctctta tcagaagata ggaaatgaaa atatcctttt ttaaaatatg tggaagtaat 2220ttgggtataa ttagtttttt tctacctttt ggaaagttgt ttttttgttg tttttttttt 2280tttcccagat gagaacatta acatagtggt taaatgtcta ggcttccatt taaaactaca 2340caaatgactt gggatctttt tagcactaag gaatttgatt tcagccttcc agctgttgct 2400gtgagttgtt ccagaccttt ctgtggcttt ttggtaaggc tgcttagaag catgagaagc 2460atgagaatgg taatgtgtgc taaacctatg tttaaccaat ctttgcacca aaggactttt 2520tcaccaattt attttgttat tcttctaaat attaagtgat ttctaaaagg taaagggtga 2580ccttttgttt ttatatctaa tttctcaatt tctttatatg catttttaga ataatttgag 2640agattaaatg ctgcttgaaa ctattatact ttgagtttta gattggccaa atacattaat 2700gtagttaaat tcatctttaa agtacacata tgtgcctaga gccaaaaaat aataatgatt 2760taatttatga ccttatgttg agactaattt cacatcttat tttacagtca tttacagtga 2820aacaatgttc cagctagctt taaaagctat acggtgctaa ttagtaaaat attgagggca 2880atattttact gctagcttgc aaaattataa gtgttttaaa aataaaatac atgaaaaaaa 2940aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 2974 23 117 PRT Homo sapiensProtein Ganglioside expression factor 2 23 Met Lys Trp Met Phe Lys GluAsp His Ser Leu Glu His Arg Cys Val 1 5 10 15 Glu Ser Ala Lys Ile ArgAla Lys Tyr Pro Asp Arg Val Pro Val Ile 20 25 30 Val Glu Lys Val Ser GlySer Gln Ile Val Asp Ile Asp Lys Arg Lys 35 40 45 Tyr Leu Val Pro Ser AspIle Thr Val Ala Gln Phe Met Trp Ile Ile 50 55 60 Arg Lys Arg Ile Gln LeuPro Ser Glu Lys Ala Ile Phe Leu Phe Val 65 70 75 80 Asp Lys Thr Val ProGln Ser Ser Leu Thr Met Gly Gln Leu Tyr Glu 85 90 95 Lys Glu Lys Asp GluAsp Gly Phe Leu Tyr Val Ala Tyr Ser Gly Glu 100 105 110 Asn Thr Phe GlyPhe 115 24 975 DNA Rattus norvegicus cDNA Ganglioside expression factor2 24 cccgcctgcc gagtagtcgt cgctgccgcc gccgcctccg ttgttgttgt ggtcgcttcg60 ccgaagtctg cggctcaaag agccggctcc gtcgcttccc gccgccatga agtggatgtt 120taaggaggac cactcgctgg aacacagatg cgtggaatcc gcgaagatca gagcgaaata 180ccccgaccgg gttccggtga tcgttgagaa agtctctggc tctcagattg ttgacattga 240caagaggaag tacttggtcc catctgacat cactgtggct cagttcatgt ggatcatcag 300gaaaaggatc cagcttcctt ctgagaaggc catcttcttg tttgtggaca agacagtccc 360acagtccagc ctaactatgg gacagcttta cgagaaggaa aaagatgaag atggattctt 420gtatgtggcc tacagcggag agaacacttt tggcttctga gcccttgctg ggctaggtgc 480acccttcctg cttgtgtatc ctgtaaataa ctggctgttc tcagttactc cgccggagcc 540tccacacaga cctactagtg catttgtaac tggatttatt tcttaatata ttggaaggtt 600ttgttttcct tagattagta aattatcata cagagtttta ttttcagttt tcttttgtgc 660actgtcctca tggctatatg ctccaaggaa cctgtcctcc ggaatcacat ttaatgaaga 720tacttccgaa atgaagggcg gtaggtgtgg tattaaagtg acaaggaggg atgacgcatt 780gttctggatt atgttcggag tgttagacgg ctaagtatta aaagccccca aattaaatcc 840ttagcaatca gaacacttgc ttcactagat tttgccaact gcaaatcatg ttggactgag 900ctaatctgtt ctttctgaga ctataaggta aatgattaac aataaagcct ccatgtaaaa 960ggcaaaaaaa aaaaa 975 25 1377 PRT Homo sapiens Protein HMG1/2 25 Ile ProMet Val Val Ser Asp Phe Asp Leu Pro Asp Gln Gln Ile Glu 1 5 10 15 IleLeu Gln Ser Ser Asp Ser Gly Cys Ser Gln Ser Ser Ala Gly Asp 20 25 30 AsnLeu Ser Tyr Glu Val Asp Pro Glu Thr Val Asn Ala Gln Glu Asp 35 40 45 SerGln Met Pro Lys Glu Ser Ser Pro Asp Asp Asp Val Gln Gln Val 50 55 60 ValPhe Asp Leu Ile Cys Lys Val Val Ser Gly Leu Glu Val Glu Ser 65 70 75 80Ala Ser Val Thr Ser Gln Leu Glu Ile Glu Ala Met Pro Pro Lys Cys 85 90 95Ser Asp Ile Asp Pro Asp Glu Glu Thr Ile Lys Ile Glu Asp Asp Ser 100 105110 Ile Gln Gln Ser Gln Asn Ala Leu Leu Ser Asn Glu Ser Ser Gln Phe 115120 125 Leu Ser Val Ser Ala Glu Gly Gly His Glu Cys Val Ala Asn Gly Ile130 135 140 Ser Arg Asn Ser Ser Ser Pro Cys Ile Ser Gly Thr Thr His ThrLeu 145 150 155 160 His Asp Ser Ser Val Ala Ser Ile Glu Thr Lys Ser ArgGln Arg Ser 165 170 175 His Ser Ser Ile Gln Phe Ser Phe Lys Glu Lys LeuSer Glu Lys Val 180 185 190 Ser Glu Lys Glu Thr Ile Val Lys Glu Ser GlyLys Gln Pro Gly Ala 195 200 205 Lys Pro Lys Val Lys Leu Ala Arg Lys LysAsp Asp Asp Lys Lys Lys 210 215 220 Ser Ser Asn Glu Lys Leu Lys Gln ThrSer Val Phe Phe Ser Asp Gly 225 230 235 240 Leu Asp Leu Glu Asn Trp TyrSer Cys Gly Glu Gly Asp Ile Ser Glu 245 250 255 Ile Glu Ser Asp Met GlySer Pro Gly Ser Arg Lys Ser Pro Asn Phe 260 265 270 Asn Ile His Pro LeuTyr Gln His Val Leu Leu Tyr Leu Gln Leu Tyr 275 280 285 Asp Ser Ser ArgThr Leu Tyr Ala Phe Ser Ala Ile Lys Ala Ile Leu 290 295 300 Lys Thr AsnPro Ile Ala Phe Val Asn Ala Ile Ser Thr Thr Ser Val 305 310 315 320 AsnAsn Ala Tyr Thr Pro Gln Leu Ser Leu Leu Gln Asn Leu Leu Ala 325 330 335Arg His Arg Ile Ser Val Met Gly Lys Asp Phe Tyr Ser His Ile Pro 340 345350 Val Asp Ser Asn His Asn Phe Arg Ser Ser Met Tyr Ile Glu Ile Leu 355360 365 Ile Ser Leu Cys Leu Tyr Tyr Met Arg Ser His Tyr Pro Thr His Val370 375 380 Lys Val Thr Ala Gln Asp Leu Ile Gly Asn Arg Asn Met Gln MetMet 385 390 395 400 Ser Ile Glu Ile Leu Thr Leu Leu Phe Thr Glu Leu AlaLys Val Ile 405 410 415 Glu Ser Ser Ala Lys Gly Phe Pro Ser Phe Ile SerAsp Met Leu Ser 420 425 430 Lys Cys Lys Val Gln Lys Val Ile Leu His CysLeu Leu Ser Ser Ile 435 440 445 Phe Ser Ala Gln Lys Trp His Ser Glu LysMet Ala Gly Lys Asn Leu 450 455 460 Val Ala Val Glu Glu Gly Phe Ser GluAsp Ser Leu Ile Asn Phe Ser 465 470 475 480 Glu Asp Glu Phe Asp Asn GlySer Thr Leu Gln Ser Gln Leu Leu Lys 485 490 495 Val Leu Gln Arg Leu IleVal Leu Glu His Arg Val Met Thr Ile Pro 500 505 510 Glu Glu Asn Glu ThrGly Phe Asp Phe Val Val Ser Asp Leu Glu His 515 520 525 Ile Ser Pro HisGln Pro Met Thr Ser Leu Gln Tyr Leu His Ala Gln 530 535 540 Pro Ile ThrCys Gln Gly Met Phe Leu Cys Ala Val Ile Arg Ala Leu 545 550 555 560 HisGln His Cys Ala Cys Lys Met His Pro Gln Trp Ile Gly Leu Ile 565 570 575Thr Ser Thr Leu Pro Tyr Met Gly Lys Val Leu Gln Arg Val Val Val 580 585590 Ser Val Thr Leu Gln Leu Cys Arg Asn Leu Asp Asn Leu Ile Gln Gln 595600 605 Tyr Lys Tyr Glu Thr Gly Leu Ser Asp Ser Arg Pro Leu Trp Met Ala610 615 620 Ser Ile Ile Pro Pro Asp Met Ile Leu Thr Leu Leu Glu Gly IleThr 625 630 635 640 Ala Ile Ile His Tyr Cys Leu Leu Asp Pro Thr Thr GlnTyr His Gln 645 650 655 Leu Leu Val Ser Val Asp Gln Lys His Leu Phe GluAla Arg Ser Gly 660 665 670 Ile Leu Ser Ile Leu His Met Ile Met Ser SerVal Thr Leu Leu Trp 675 680 685 Ser Ile Leu His Gln Ala Asp Ser Ser GluLys Met Thr Ile Ala Ala 690 695 700 Ser Ala Ser Leu Thr Thr Ile Asn LeuGly Ala Thr Lys Asn Leu Arg 705 710 715 720 Gln Gln Ile Leu Glu Leu LeuGly Pro Ile Ser Met Asn His Gly Val 725 730 735 His Phe Met Ala Ala IleAla Phe Val Trp Asn Glu Arg Arg Gln Asn 740 745 750 Lys Thr Thr Thr ArgThr Lys Val Ile Pro Ala Ala Ser Glu Glu Gln 755 760 765 Leu Leu Leu ValGlu Leu Val Arg Ser Ile Ser Val Met Arg Ala Glu 770 775 780 Thr Val IleGln Thr Val Lys Glu Val Leu Lys Gln Pro Pro Ala Ile 785 790 795 800 AlaLys Asp Lys Lys His Leu Ser Leu Glu Val Cys Met Leu Gln Phe 805 810 815Phe Tyr Ala Tyr Ile Gln Arg Ile Pro Val Pro Asn Leu Val Asp Ser 820 825830 Trp Ala Ser Leu Leu Ile Leu Leu Lys Asp Ser Ile Gln Leu Ser Leu 835840 845 Pro Ala Pro Gly Gln Phe Leu Ile Leu Gly Val Leu Asn Glu Phe Ile850 855 860 Met Lys Asn Pro Ser Leu Glu Asn Lys Lys Asp Gln Arg Asp LeuGln 865 870 875 880 Asp Val Thr His Lys Ile Val Asp Ala Ile Gly Ala IleAla Gly Ser 885 890 895 Ser Leu Glu Gln Thr Thr Trp Leu Arg Arg Asn LeuGlu Val Lys Pro 900 905 910 Ser Pro Lys Ile Met Val Asp Gly Thr Asn LeuGlu Ser Asp Val Glu 915 920 925 Asp Met Leu Ser Pro Ala Met Glu Thr AlaAsn Ile Thr Pro Ser Val 930 935 940 Tyr Ser Val His Ala Leu Thr Leu LeuSer Glu Val Leu Ala His Leu 945 950 955 960 Leu Asp Met Val Phe Tyr SerAsp Glu Lys Glu Arg Val Ile Pro Leu 965 970 975 Leu Val Asn Ile Met HisTyr Val Val Pro Tyr Leu Arg Asn His Ser 980 985 990 Ala His Asn Ala ProSer Tyr Arg Ala Cys Val Gln Leu Leu Ser Ser 995 1000 1005 Leu Ser GlyTyr Gln Tyr Thr Arg Arg Ala Trp Lys Lys Glu Ala 1010 1015 1020 Phe AspLeu Phe Met Asp Pro Ser Phe Phe Gln Met Asp Ala Ser 1025 1030 1035 CysVal Asn His Trp Arg Ala Ile Met Asp Asn Leu Met Thr His 1040 1045 1050Asp Lys Thr Thr Phe Arg Asp Leu Met Thr Arg Val Ala Val Ala 1055 10601065 Gln Ser Ser Ser Leu Asn Leu Phe Ala Asn Arg Asp Val Glu Leu 10701075 1080 Glu Gln Arg Ala Met Leu Leu Lys Arg Leu Ala Phe Ala Ile Phe1085 1090 1095 Ser Ser Glu Ile Asp Gln Tyr Gln Lys Tyr Leu Pro Asp IleGln 1100 1105 1110 Glu Arg Leu Val Glu Ser Leu Arg Leu Pro Gln Val ProThr Leu 1115 1120 1125 His Ser Gln Val Phe Leu Phe Phe Arg Val Leu LeuLeu Arg Met 1130 1135 1140 Ser Pro Gln His Leu Thr Ser Leu Trp Pro ThrMet Ile Thr Glu 1145 1150 1155 Leu Val Gln Val Phe Leu Leu Met Glu GlnGlu Leu Thr Ala Asp 1160 1165 1170 Glu Asp Ile Ser Arg Thr Ser Gly ProSer Val Ala Gly Leu Glu 1175 1180 1185 Thr Thr Tyr Thr Gly Gly Asn GlyPhe Ser Thr Ser Tyr Asn Ser 1190 1195 1200 Gln Arg Trp Leu Asn Leu TyrLeu Ser Ala Cys Lys Phe Leu Asp 1205 1210 1215 Leu Ala Leu Ala Leu ProSer Glu Asn Leu Pro Gln Phe Gln Met 1220 1225 1230 Tyr Arg Trp Ala PheIle Pro Glu Ala Ser Asp Asp Ser Gly Leu 1235 1240 1245 Glu Val Arg ArgGln Gly Ile His Gln Arg Glu Phe Lys Pro Tyr 1250 1255 1260 Val Val ArgLeu Ala Lys Leu Leu Arg Lys Arg Ala Lys Lys Asn 1265 1270 1275 Pro GluGlu Asp Asn Ser Gly Arg Thr Leu Gly Trp Glu Pro Gly 1280 1285 1290 HisLeu Leu Leu Thr Ile Cys Thr Val Arg Ser Met Glu Gln Leu 1295 1300 1305Leu Pro Phe Phe Asn Val Leu Ser Gln Val Phe Asn Ser Lys Val 1310 13151320 Thr Ser Arg Cys Gly Gly His Ser Gly Ser Pro Ile Leu Tyr Ser 13251330 1335 Asn Ala Phe Pro Asn Lys Asp Met Lys Leu Glu Asn His Lys Pro1340 1345 1350 Cys Ser Ser Lys Ala Arg Gln Lys Ile Glu Glu Met Val GluLys 1355 1360 1365 Asp Phe Leu Glu Gly Met Ile Lys Thr 1370 1375 26 1000DNA Homo sapiens cDNA HMG1/2 26 tttttttttt tttttttttg aaaaaaaaaatgggtagtgt atattttgca ggtttaagac 60 aactcaggac aataaaaaca atggactttacatgtgtata tatatagctc tcttaggcac 120 cataatcagt atgagccaac aatatttaaacttgattcag gccacattca gacatttgct 180 cttatataca aatatttaaa ttaaatacaatctgaaatgt gttctgttac atacaaaaaa 240 ggaaaaacta tacaacgcag agcagtgtgtgtgttttaaa taattacatt tacatgtaag 300 ctaaatggaa ccagcaatgg tgctcaagtttttatcatcc cttccagaaa atctttttct 360 accatctctt ctattttttg cctggctttgctggaacatg gtttgtggtt ctccagtttc 420 atgtccttat tagggaaggc atttgagtagaggataggac tccctgagtg tcctccacat 480 cggcttgtga ctttgctgtt gaagacttgactgagcacat tgaagaacgg caggagctgc 540 tccatactgc gcacggtgca gatggtgagcagcaagtgcc ctggctccca acccaatgtt 600 ctccctgagt tgtcttcctc tggatttttctttgctcttt tccgaagaag ttttgctagt 660 cgtaccacgt aaggtttaaa ttctcgttgatgtataccct gccttctgac ttccaaacct 720 gaatcatctg aggcttctgg aataaaggcccatcggtaca tctgaaactg aggaaggttt 780 tcagagggca atgcgagagc caaatccaaaaatttgcaag cagagagata gaggtttaac 840 caccgctggc tgttatatga agtagagaagccattacctc ctgtgtacgt tgtctccaga 900 ccagccacag agggccctga agtccgtgaaatatcttcat cagcagtgag ttcctgctcc 960 atcagtaaaa atacttgtac aagttctgtaatcatggtag 1000 27 896 PRT Mus musculus Protein Mouse phosphoprotein(F1-20) 27 Met Ser Gly Gln Thr Leu Thr Asp Arg Ile Ala Ala Ala Gln TyrSer 1 5 10 15 Val Thr Gly Ser Ala Val Ala Arg Ala Val Cys Lys Ala ThrThr His 20 25 30 Glu Val Met Gly Pro Lys Lys Lys His Leu Asp Tyr Leu IleGln Ala 35 40 45 Thr Asn Glu Thr Asn Val Asn Ile Pro Gln Met Ala Asp ThrLeu Phe 50 55 60 Glu Arg Ala Thr Asn Ser Ser Trp Val Val Val Phe Lys AlaLeu Val 65 70 75 80 Thr Thr His His Leu Met Val His Gly Asn Glu Arg PheIle Gln Tyr 85 90 95 Leu Ala Ser Arg Asn Thr Leu Phe Asn Leu Ser Asn PheLeu Asp Lys 100 105 110 Ser Gly Ser His Gly Tyr Asp Met Ser Thr Phe IleArg Arg Tyr Ser 115 120 125 Arg Tyr Leu Asn Glu Lys Ala Phe Ser Tyr ArgGln Met Ala Phe Asp 130 135 140 Phe Ala Arg Val Lys Lys Gly Ala Asp GlyVal Met Arg Thr Met Val 145 150 155 160 Pro Glu Lys Leu Leu Lys Ser MetPro Ile Leu Gln Gly Gln Ile Asp 165 170 175 Ala Leu Leu Glu Phe Asp ValHis Pro Asn Glu Leu Thr Asn Gly Val 180 185 190 Ile Asn Ala Ala Phe MetLeu Leu Phe Lys Asp Leu Ile Lys Leu Phe 195 200 205 Ala Cys Tyr Asn AspGly Val Ile Asn Leu Leu Glu Lys Phe Phe Glu 210 215 220 Met Lys Lys GlyGln Cys Lys Asp Ala Leu Glu Ile Tyr Lys Arg Phe 225 230 235 240 Leu ThrArg Met Thr Arg Val Ser Glu Phe Leu Lys Val Ala Glu Gln 245 250 255 ValGly Ile Asp Lys Gly Asp Ile Pro Asp Leu Thr Gln Ala Pro Ser 260 265 270Ser Leu Met Glu Thr Leu Glu Gln His Leu Asn Thr Leu Glu Gly Lys 275 280285 Lys Pro Gly Asn Asn Glu Gly Ser Gly Ala Pro Ser Pro Leu Ser Lys 290295 300 Ser Ser Pro Ala Thr Thr Val Thr Ser Pro Asn Ser Thr Pro Ala Lys305 310 315 320 Thr Ile Asp Thr Ser Pro Pro Val Asp Ile Phe Ala Thr AlaSer Ala 325 330 335 Ala Ala Pro Val Ser Ser Ala Lys Pro Ser Ser Asp LeuLeu Asp Leu 340 345 350 Gln Pro Asp Phe Ser Gly Ala Ala Ala Gly Ala AlaAla Pro Val Val 355 360 365 Pro Pro Ser Gly Gly Ala Thr Ala Trp Gly AspLeu Leu Gly Glu Asp 370 375 380 Ser Leu Ala Ala Leu Ser Ser Val Pro CysGlu Ala Pro Ile Ser Asp 385 390 395 400 Pro Phe Ala Pro Glu Pro Ser ProPro Thr Thr Thr Thr Glu Pro Ala 405 410 415 Ser Ala Ser Ala Ser Thr ThrThr Ala Val Thr Ala Val Thr Thr Glu 420 425 430 Val Asp Leu Phe Gly AspAla Phe Ala Ala Ser Pro Gly Glu Ala Pro 435 440 445 Ala Ala Ser Glu GlyAla Thr Ala Pro Ala Thr Pro Ala Pro Val Ala 450 455 460 Ala Ala Leu AspAla Cys Ser Gly Asn Asp Pro Phe Ala Pro Ser Glu 465 470 475 480 Gly SerAla Glu Ala Ala Pro Glu Leu Asp Leu Phe Ala Met Lys Pro 485 490 495 ProGlu Thr Ser Ala Pro Val Val Thr Pro Thr Ala Ser Thr Ala Pro 500 505 510Pro Val Pro Ala Thr Ala Pro Ser Pro Ala Pro Thr Ala Val Ala Ala 515 520525 Thr Ala Ala Thr Thr Thr Ala Ala Ala Ala Ala Thr Thr Thr Ala Thr 530535 540 Thr Ser Ala Ala Ala Ala Thr Thr Ala Ala Ala Pro Pro Ala Leu Asp545 550 555 560 Ile Phe Gly Asp Leu Phe Asp Ser Ala Pro Glu Val Ala AlaAla Pro 565 570 575 Lys Pro Asp Ala Ala Pro Ser Ile Asp Leu Phe Gly ThrAsp Ala Phe 580 585 590 Ser Ser Pro Pro Arg Gly Ala Ser Pro Val Pro GluSer Ser Leu Thr 595 600 605 Ala Asp Leu Leu Ser Val Asp Ala Phe Ala AlaPro Ser Pro Ala Ser 610 615 620 Thr Ala Ser Pro Ala Lys Ala Glu Ser SerGly Val Ile Asp Leu Phe 625 630 635 640 Gly Asp Ala Phe Gly Ser Gly AlaSer Glu Thr Gln Pro Ala Pro Gln 645 650 655 Ala Val Ser Ser Ser Ser AlaSer Ala Asp Leu Leu Ala Gly Phe Gly 660 665 670 Gly Ser Phe Met Ala ProSer Thr Thr Pro Val Thr Pro Ala Gln Asn 675 680 685 Asn Leu Leu Gln ProSer Phe Glu Ala Ala Phe Gly Thr Thr Pro Ser 690 695 700 Thr Ser Ser SerSer Ser Phe Asp Pro Ser Gly Asp Leu Leu Met Pro 705 710 715 720 Thr MetAla Pro Ser Gly Gln Pro Ala Pro Val Ser Met Val Pro Pro 725 730 735 SerPro Ala Met Ala Ala Ser Lys Gly Leu Gly Ser Asp Leu Asp Ser 740 745 750Ser Leu Ala Ser Leu Val Gly Asn Leu Gly Ile Ser Gly Thr Thr Ser 755 760765 Lys Lys Gly Asp Leu Gln Trp Asn Ala Gly Glu Lys Lys Leu Thr Gly 770775 780 Gly Ala Asn Trp Gln Pro Lys Val Thr Pro Ala Thr Trp Ser Ala Gly785 790 795 800 Val Pro Pro Gln Gly Thr Val Pro Pro Thr Ser Ser Val ProPro Gly 805 810 815 Ala Gly Ala Pro Ser Val Gly Gln Pro Gly Ala Gly PheGly Met Pro 820 825 830 Pro Ser Gly Thr Gly Met Thr Met Met Ser Gln GlnPro Val Met Phe 835 840 845 Ala Gln Pro Met Met Arg Pro Pro Phe Gly AlaAla Ala Val Pro Gly 850 855 860 Thr Gln Leu Ser Pro Ser Pro Thr Pro AlaThr Gln Ser Pro Lys Lys 865 870 875 880 Pro Pro Ala Lys Asp Pro Leu AlaAsp Leu Asn Ile Lys Asp Phe Leu 885 890 895 28 4260 DNA Mus musculuscDNA Mouse phosphoprotein (F1-20) 28 gggacagggg ccgggccggg gctacaggcatcgcggcccg gggacaccag ggcggtgcgt 60 gtctgcaccc agctacctcg cgcggcgtccgggctgcggt gctcctgggg cgggaagagg 120 aggcggtaga cgcggccggt gaagatgtcgggccaaacgc tcacggatcg gatcgccgcc 180 gctcagtaca gcgtgactgg ctctgctgtagcaagagcag tctgcaaagc caccactcat 240 gaggtgatgg gccccaagaa gaagcacctggactatttga tccaggctac caatgagacc 300 aatgtcaata tccctcagat ggccgacaccctctttgagc gggcgacaaa cagtagctgg 360 gtggtggtat ttaaagcttt agtgaccacacaccatctca tggtgcatgg aaatgagaga 420 tttattcagt atttggcctc taggaatacgctattcaatc tcagcaactt tctagataaa 480 agtggatccc acggttatga tatgtctacgttcatacggc gttacagtag atacttgaat 540 gaaaaggctt tctcctacag acagatggcatttgactttg ccagagtgaa gaaaggggct 600 gacggtgtca tgaggacgat ggttcctgaaaagctcctga agagtatgcc aatcctgcag 660 gggcagatcg atgcactgct ggagtttgatgtgcatccaa atgaactaac caatggtgtc 720 ataaatgctg catttatgct tcttttcaaagatcttatca aactgtttgc ttgctacaat 780 gacggcgtca ttaacttact tgaaaaattttttgagatga agaaaggtca atgcaaagac 840 gcgctagaaa tttacaagcg atttctaactagaatgacga gggtgtccga attcctcaag 900 gtcgccgagc aagttggtat tgataaaggcgacattcccg acctcacgca ggctcccagc 960 agtcttatgg agacccttga acaacatctaaataccctag aaggaaagaa acctggaaac 1020 aatgaaggat ctggtgctcc ctctccactaagtaagtctt ctccagccac aactgttaca 1080 tctcctaatt ctacaccagc taaaactatcgacacatccc cgccagttga catatttgca 1140 acagcatccg cggctgcccc agtcagctctgctaagccat caagcgatct ccttgatctt 1200 cagcccgact tctctggagc ggctgcgggggcagcagcac ctgtagtgcc tccttctggg 1260 ggtgcgaccg cctggggaga ccttttgggagaggattcct tggctgcact ttcctctgtt 1320 ccctgtgaag caccgatttc agacccatttgcaccagagc cttcccctcc tactacaacc 1380 actgagcctg cttcagcctc tgcctcgaccaccacagctg tgacggctgt cactacggaa 1440 gtggatctct ttggagatgc ctttgcagcttctcctgggg aggcccctgc agcatccgaa 1500 ggggctaccg caccagctac cccggccccagtggctgcag ctcttgatgc atgctcagga 1560 aatgaccctt ttgccccatc tgaaggtagcgcagaggctg cacctgagct ggacctcttt 1620 gcaatgaagc cacctgagac cagcgctcctgtagttaccc ctacagctag cacagcccct 1680 ccagttcccg caactgctcc ttctcctgctcccacggctg tggcagccac tgctgccacc 1740 accaccgccg ccgctgcagc taccaccactgccaccacct ctgctgctgc tgccaccacc 1800 gccgctgctc ctcctgctct agatatctttggtgatttgt ttgattctgc tcctgaagtt 1860 gctgcagcac ctaagccaga cgcggctcctagcatagacc tgtttggcac agatgctttc 1920 tcctccccgc cacgaggggc ctctccggtgcctgagagtt ctctcactgc tgacctctta 1980 tctgtggacg catttgcagc gccgtctcctgcatccactg cctctcctgc aaaggcggag 2040 tcctcgggtg tcatagacct ttttggggatgcgtttggaa gtggtgcttc tgaaacccag 2100 ccagcaccac aggctgtttc tagttcatcagcatcggcag atctactagc tggatttggg 2160 ggttctttca tggccccttc tacaacgccagtgactccag ctcagaataa cctgctgcaa 2220 cccagtttcg aggcagcttt tggaacgacgccttcgactt caagcagcag ctcttttgac 2280 ccatcagtgt ttgatggttt aggcgatcttctgatgccaa ccatggcacc atccgggcag 2340 cctgcccctg tctcaatggt cccacccagtcctgcaatgg cagccagcaa aggcctcgga 2400 agtgaccttg actcgtctct ggccagtttagtaggcaatc ttgggatttc tggtaccaca 2460 tcaaaaaagg gagatctcca gtggaatgctggggagaaaa agctgactgg tggagccaac 2520 tggcaaccga aagtcactcc agccacatggtcagcgggtg ttcccccgca aggcactgtt 2580 ccaccaacca gctcagtccc tccgggtgccggggccccgt ccgttgggca acctggagca 2640 ggatttggaa tgcctccttc agggacaggcatgaccatga tgtctcagca gccagtcatg 2700 tttgcacagc ccatgatgag gccaccctttggagctgcag ctgtgcccgg cacacagctt 2760 tctccaagcc ctacacctgc cactcagagtcccaagaaac ctccagccaa ggacccgtta 2820 gcggatctta acatcaagga tttcttgtaaacaatttaag ctgcaatttt tgtgactgaa 2880 taggaaaaaa ctaacctgag tttggaaacttcagataaga ttgatgctca gtttcaaagt 2940 gagccaccag taccaaaccc agtgtgacgcgtaacttcct ctcccaagca cacaggccag 3000 ctgtggcagt gaacattagg aatgtgtactctttagctgt taccctgctc ttccagctcg 3060 tagcgtattc gggttctttg tgtgttgtacgaagtaaacc atgagtgaat gaatggttcc 3120 aatgccttta gtgcttttct ggacgttgcccgtggacgga ggatgacgca gctgttctgt 3180 ggtgagccat ttggaaagat gtgtctgtgttttcgaaagg tttcgatgta tatataactt 3240 ttggacaaac tcaagtcctc ccatgaactttctcttctct tctgtacctc tgttacaagc 3300 gtaatgtgat actatcagct agtgagacaaacactcttaa ctatacaaaa actttttcgg 3360 tgtggagtac atttttccaa tcacagaaacttccaacttg ttgtgagaaa tgtttatttt 3420 tgtgcactgt atatgttaag aaattttattttaaaaaaaa atgaagtcta atgtccataa 3480 taaaacttct ccttgatgaa gctaccttatcaagaatgag aaaaaccata tgagaagtcc 3540 atatttatca ctgctatatt aagatatatattttaattat atttgcaggg tttgcatcta 3600 aattgaccta tttattcatt tgtgatcaaaggcaccgaaa agtggagctt gtttctattg 3660 tgtctgtgac aatgcagtta gagatgtgctgtttctgtga ctacgaacgt gccccaagag 3720 acatctgtaa cttaaagaga actgcaaatattttttattt cagtgtgggt ttacgtacat 3780 ctgttcagtt agtatttctt tgtgtgttctatagagtagt gtttctccat ccttcaattg 3840 agctcaaagt gacttctatt gtacctttgtgataggattg aaaccaattc agcgaattgt 3900 atcttttaat gtacataatg tattctttgattttcagttt gttgtcatac tgtctgtgcc 3960 gatggcttgg ctgaagattt tgatgcgtacacaaggtcac tgttgatcag tgttgtttag 4020 tggcttggca gctctttgta aaagcatattgggttggaaa ggcatttgcc tatttttcaa 4080 attatttaat agatgtatgg taccctttaaagtggttgta tctgaattta ctgtggggat 4140 aacatacact gtaatggggg aaaattacctaaaaccaatt tcaaaatggc ttttctttgt 4200 attttggttt aaaaacccag tgcatgtctgccctctgaga tgcaataaac accttgaacc 4260 29 196 PRT Rattus norvegicusProtein ribosomal protein L 19 29 Met Ser Met Leu Arg Leu Gln Lys ArgLeu Ala Ser Ser Val Leu Arg 1 5 10 15 Cys Gly Lys Lys Lys Val Trp LeuAsp Pro Asn Glu Thr Asn Glu Ile 20 25 30 Ala Asn Ala Asn Ser Arg Gln GlnIle Arg Lys Leu Ile Lys Asp Gly 35 40 45 Leu Ile Ile Arg Lys Pro Val ThrVal His Ser Arg Ala Arg Cys Arg 50 55 60 Lys Asn Thr Leu Ala Arg Arg LysGly Arg His Met Gly Ile Gly Lys 65 70 75 80 Arg Lys Gly Thr Ala Asn AlaArg Met Pro Glu Lys Val Thr Trp Met 85 90 95 Arg Arg Met Arg Ile Leu ArgArg Leu Leu Arg Arg Tyr Arg Glu Ser 100 105 110 Lys Lys Ile Asp Arg HisMet Tyr His Ser Leu Tyr Leu Lys Val Lys 115 120 125 Gly Asn Val Phe LysAsn Lys Arg Ile Leu Met Glu His Ile His Lys 130 135 140 Leu Lys Ala AspLys Ala Arg Lys Lys Leu Leu Ala Asp Gln Ala Glu 145 150 155 160 Ala ArgArg Ser Lys Thr Lys Glu Ala Arg Lys Arg Arg Glu Glu Arg 165 170 175 LeuGln Ala Lys Lys Glu Glu Ile Ile Lys Thr Leu Ser Lys Glu Glu 180 185 190Glu Thr Lys Lys 195 30 701 DNA Rattus norvegicus cDNA ribosomal proteinL 19 30 ctttcctttc gctgctgcgt ctgcagccat gagtatgctt aggctacagaagaggcttgc 60 ctctagcgtc ctccgctgtg gtaaaaagaa ggtgtggttg gaccccaatgaaaccaacga 120 aatcgccaat gccaactctc gtcaacagat caggaagctg atcaaagatggcctgatcat 180 ccggaagcct gtgactgtcc attcccgggc tcgatgccgg aagaacaccttggcccgacg 240 gaagggcagg catatgggca tagggaagag gaagggtact gccaacgctcggatgcccga 300 gaaggtgacc tggatgcgaa ggatgaggat cctgcgccgg cttctcaggagataccggga 360 atctaagaag attgaccgtc atatgtatca cagcctgtac ctgaaggtcaaagggaatgt 420 gttcaaaaac aagcggattc tcatggagca catccacaaa ctgaaggcagacaaggcccg 480 caagaagcta ctggctgacc aggctgaggc tcgcaggtct aagaccaaggaagcacgaaa 540 gcgccgggag gagcgcctcc aagccaagaa ggaggagatc atcaagactctgtccaagga 600 ggaagagacc aagaaatgaa gcgtccctcg tgtctgtaca tagtggctaggctatggccc 660 acatggatca gtcattaaaa taaaacaagc cttcgtcctt g 701 31 245PRT Rattus norvegicus Protein 14-3-3 zeta-isoform 31 Met Asp Lys Asn GluLeu Val Gln Lys Ala Lys Leu Ala Glu Gln Ala 1 5 10 15 Glu Arg Tyr AspAsp Met Ala Ala Cys Met Lys Ser Val Thr Glu Gln 20 25 30 Gly Ala Glu LeuSer Asn Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr 35 40 45 Lys Asn Val ValGly Ala Arg Arg Ser Ser Trp Arg Val Val Ser Ser 50 55 60 Ile Glu Gln LysThr Glu Gly Ala Glu Lys Lys Gln Gln Met Ala Arg 65 70 75 80 Glu Tyr ArgGlu Lys Ile Glu Met Glu Leu Arg Asp Ile Cys Asn Asp 85 90 95 Val Leu SerLeu Leu Glu Lys Phe Leu Ile Pro Asn Ala Ser Gln Pro 100 105 110 Glu SerLys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Tyr Arg Tyr 115 120 125 LeuAla Glu Val Ala Ala Gly Asp Asp Lys Lys Gly Ile Val Asp Gln 130 135 140Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile Ser Lys Lys Glu Met 145 150155 160 Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe Ser Val165 170 175 Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Glu Lys Ala Cys Ser LeuAla 180 185 190 Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr LeuSer Glu 195 200 205 Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu LeuArg Asp Asn 210 215 220 Leu Thr Leu Trp Thr Ser Asp Thr Gln Gly Asp GluAla Glu Ala Gly 225 230 235 240 Glu Gly Gly Glu Asn 245 32 1687 DNARattus norvegicus cDNA 14-3-3 zeta-isoform 32 cccagagact gccgagccgggtccgtgtgc cgccacccac tccggacaca gaatatccag 60 ttatggataa aaatgagctggtgcagaagg ccaagctggc cgagcaggca gagcgatacg 120 atgacatggc agcctgcatgaagtctgtca ctgagcaagg agccgagctg tctaacgagg 180 agaggaacct tctctctgttgcttataaaa acgttgtagg agcccgtagg tcatcttgga 240 gggtcgtctc gagtattgagcagaagacgg aaggtgctga gaaaaagcag cagatggctc 300 gagaatacag agagaagatcgagatggagc tgagggacat ctgcaacgac gtactgtctc 360 ttttggaaaa gttcttgatccccaatgctt cgcagccaga aagcaaagtc ttctatttga 420 aaatgaaggg tgactactaccgctacttgg ctgaggttgc tgctggtgat gacaagaaag 480 gaattgtgga ccagtcacagcaagcatacc aagaagcatt tgaaatcagc aaaaaggaga 540 tgcagccgac acaccccatcagactgggtc tggccctcaa cttctctgtg ttctactatg 600 agatcctgaa ctccccagagaaagcctgct ctcttgcaaa aacagctttt gatgaagcca 660 ttgctgaact tgatacattaagtgaagagt cgtacaaaga cagcacgcta ataatgcagt 720 tactgagaga caacttgacattgtggacat cggataccca aggagacgaa gcagaagcgg 780 gagaaggagg ggaaaattaaccggccttcc aacctttgtc tgcctcattc taaaatttac 840 acagtagacc atttgtcatccatgctgtcc cacagatagt ttttttgttt acgatttatg 900 acaggtttat gttacttctatttgaatttc tatatttccc atgtggtttt atgttttaat 960 attaggggag tagagccagttaactttagg gagttactcg ttttcatctt gaggtggcca 1020 atatgggatg tggaatttttacatgagtta cacatgtttg gcatagttag tacttttggt 1080 ccattgtggc ttcagaagggccagtgttca aactgcttcc atgtctaagc aaagaaaact 1140 gcctacatat tggtgtgtgctggcggggaa taaacaggat aatgggtcca gtcatgagtg 1200 tagtctgtgt gggtactgtaaggcttggag cacttgtgag gctgggacac gaacaccctg 1260 tggatgcacg ctaagaccgtgtgtctgcgt gcacaccctt gaccacagct ccagaagttg 1320 tctgtagaca aagttgtgacccaatttact ctgataaggg cagaaacggt tcacattcca 1380 ttatttgtaa agttacctgctgtttgcttt cattattttt gctacacatt ttatttgtat 1440 ttaaatgttt taggcaatctaagaacaaat gtaaaagtaa agatgcagta caaacgagtt 1500 gcttggtgtt cccggctccatgcggatcaa gcacagcggt aaacaaaatc ccatgtattt 1560 aacttttttt ttttaagttttttgttttgt tttgtttttg cttttgtgat tttttttctc 1620 tttttgatac ttgcctaacatgcatgtgct gtaaaaatag ttaacaggga aataacttga 1680 gatgacg 1687 33 246 PRTRattus norvegicus Protein 14-3-3-protein eta subtype 33 Met Gly Asp ArgGlu Gln Leu Leu Gln Arg Ala Arg Leu Ala Glu Gln 1 5 10 15 Ala Glu ArgTyr Asp Asp Met Ala Ser Ala Met Lys Ala Val Thr Glu 20 25 30 Leu Asn GluPro Leu Ser Asn Glu Asp Arg Asn Leu Leu Ser Val Ala 35 40 45 Tyr Lys AsnVal Val Gly Ala Arg Arg Ser Ser Trp Arg Val Ile Ser 50 55 60 Ser Ile GluGln Lys Thr Met Ala Asp Gly Asn Glu Lys Lys Leu Glu 65 70 75 80 Lys ValLys Ala Tyr Arg Glu Lys Ile Glu Lys Glu Leu Glu Thr Val 85 90 95 Cys AsnAsp Val Leu Ala Leu Leu Asp Lys Phe Leu Ile Lys Asn Cys 100 105 110 AsnAsp Phe Gln Tyr Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly 115 120 125Asp Tyr Tyr Arg Tyr Leu Ala Glu Val Ala Ser Gly Glu Lys Lys Asn 130 135140 Ser Val Val Glu Ala Ser Glu Ala Ala Tyr Lys Glu Ala Phe Glu Ile 145150 155 160 Ser Lys Glu His Met Gln Pro Thr His Pro Ile Arg Leu Gly LeuAla 165 170 175 Leu Asn Phe Ser Val Phe Tyr Tyr Glu Ile Gln Asn Ala ProGlu Gln 180 185 190 Ala Cys Leu Leu Ala Lys Gln Ala Phe Asp Asp Ala IleAla Glu Leu 195 200 205 Asp Thr Leu Asn Glu Asp Ser Tyr Lys Asp Ser ThrLeu Ile Met Gln 210 215 220 Leu Leu Arg Asp Asn Leu Thr Leu Trp Thr SerAsp Gln Gln Asp Glu 225 230 235 240 Glu Ala Gly Glu Gly Asn 245 34 1689DNA Rattus norvegicus cDNA 14-3-3-protein eta subtype 34 tgcagccagctagcgagaag gcgcgagcgg cggcgcagcc agcagcctcc cgccagccgg 60 cgagccagtgcgcgtgcgcg gcggcggcct cggcggcgac cgggaagcgg acgggcgggc 120 gaggcgagcgaggcaggcgg tgcgggcgtg cgaggcgagg ccgatcgcga gcgacatggg 180 ggaccgagagcagctgctgc agcgggcgcg actggcggag caggcggagc gctacgacga 240 catggcctccgccatgaagg cggtgacaga gctgaatgaa cctctatcta atgaagatag 300 aaatctcctctctgtggcct acaagaatgt agttggtgcc aggcgatctt cttggagggt 360 tattagtagcattgagcaga aaaccatggc agatgggaat gagaagaagc tggagaaagt 420 caaagcctatcgggagaaga ttgagaagga gctggagaca gtttgcaatg atgtcttggc 480 tctgctcgacaagttcctta tcaagaactg caatgatttt cagtacgaga gcaaggtgtt 540 ctacctgaaaatgaagggcg attactaccg ctacctggca gaggtggctt ctggggagaa 600 gaaaaacagtgtggttgaag cttctgaggc agcgtataag gaagccttcg aaatcagcaa 660 agagcacatgcagccaacac accccatccg gcttggcctg gccctcaatt tttctgtgtt 720 ctactatgagatccagaatg caccagagca ggcctgcctc ttagccaaac aagccttcga 780 tgatgctatagctgagctgg acacattaaa cgaggattcc tataaggact ccactctcat 840 catgcagttgctgcgagaca acctcaccct ctggacgagc gaccagcagg atgaagaagc 900 cggagaaggcaactgaagac ccatcaggtc cctggccctt cctttaccca ccacccccat 960 tatcactgattcttccttgc cacaatcact atatctagtg ctaaacctat ctgtattggc 1020 agcacagctattcagatctg ccctcctgtc ccttggaagc agtttcagat aaaccttcat 1080 gggcatttgctggactgatg gttgctttga gccacagagc gctccctttt tgaattgtgc 1140 agagaagtgtgttctgaacg aggcatttta ttatgtctgt tgatctgtag caaatccatg 1200 tgatggtaattgagtgtaga aaggagaatt agccaacaca ggctatggct gctatttaaa 1260 acaagctgatagtgtgttgt taagcagtac atctcgtgca tgcaaaaatg aatttgaccc 1320 tctcaccccttctttcagct aatggaaact gacacacgac aacttgttcc ttcaccatca 1380 gctttataaactgtttctcg tgagctttca ggcccctgct gtgcctcttt aaattatgat 1440 gtgcgcacaccttcttttca atgcaatgca tcagaggttt ttgatatgtg taactttttt 1500 ttttggttgtgattaagaat catggattta ttttttgtaa ctctttggct attgttcttg 1560 tgtaccctgacagcatcatg tgtgtcaacc tgtgtcaatc atgatgggtg gttatgaaat 1620 gccagattgctaaaataaat gttttggact taaaaagagt aaataaatgc tgctttgggg 1680 atattaaaa1689

1. Use of: (a) an isolated gene sequence that is up-regulated in thespinal cord of a mammal in response to mechanistically distinct firstand second models of neuropathic or central sensitization pain; (b) anisolated gene sequence comprising a nucleic acid sequence of Table 1;(c) an isolated gene sequence having at least 80% sequence identity witha nucleic acid sequence of Table 1; (d) an isolated nucleic acidsequence that is hybridizable to any of the gene sequences according to(a), (b) or (c) under stringent hybridisation conditions; (e) arecombinant vector comprising a gene sequence or nucleic acid sequenceaccording to any one of (a) to (d); (f) a host cell comprising thevector according to (e); (g) a non-human animal having in its genome anintroduced gene sequence or nucleic acid sequence or a removed ordown-regulated gene sequence or nucleic acid sequence according to anyone of (a) to (d); (h) an isolated polypeptide comprising an amino acidsequence at least 90% identical to an amino acid sequence encoded by anucleotide sequence according to any one of (a) to (d), or a polypeptidevariant thereof with sequential amino acid deletions from the C terminusand/or the N-terminus; (i) an isolated polypeptide encoded by anucleotide sequence according to any one of (a) to (d); or (j) anisolated antibody that binds specifically to a polypeptide according to(h) or (i); in the screening of compounds for the treatment of pain, orfor the diagnosis of pain.
 2. Use according to claim 1, wherein theisolated gene sequence is up-regulated both in response tostreptozocin-induced diabetes and in response to surgical injury of anerve leading to the spine.
 3. Use according to claim 1 wherein theisolated gene sequence encodes a kinase.
 4. Use according to claim 3,wherein the isolated gene sequence encodes an expression product orfragment thereof of phosphofructokinase, muscle (PFK-M) (U25651,AF249894, Y00698).
 5. Use according to claim 1, wherein the isolatedgene sequence encodes an expression product or fragment thereof of A-rafoncogene, liver expressed (X06942, U01337).
 6. Use according to claim 1wherein the isolated gene sequence encodes a phosphatase.
 7. Useaccording to claim 1, wherein the isolated gene sequence encodes anexpression product or fragment thereof of protein phosphatase I (D90164,X80910).
 8. Use according to claim 1 wherein the isolated gene sequenceencodes a phosphodiesterase.
 9. Use according to claim 8, wherein theisolated gene sequence encodes an expression product or fragment thereofof alkaline phosphodiesterase I ((D28560, AF123542, D45421).
 10. Useaccording to claim 1 wherein the isolated gene sequence encodes an ionchannel protein.
 11. Use according to claim 10, wherein the ion channelprotein is putative vacuolar ATP synthase subunit A (U13837, AF113129)or Na⁺ K⁺ ATPase alpha+isoform catalytic subunit (M14512).
 12. Useaccording to claim 1 wherein the isolated gene sequence encodes a DNAbinding protein.
 13. Use according to claim 12, wherein the isolatedgene sequence encodes an expression product or fragment thereof ofhypoxia-inducible factor-i alpha (Hif1a) (AF057308, AF003695, U22431).14. Use according to claim 1 wherein the isolated gene sequence encodesan oxidoreductase or hydrolase.
 15. Use according to claim 14, whereinthe isolated gene sequence encodes an expression product or fragmentthereof of cytochrome-c oxidase II, mitochondrial (M64496) or roundspermatid protein RSP29 (U97667).
 16. A non-human animal having in itsgenome an introduced gene sequence or a removed or down-regulated genesequence, said sequence being up-regulated in the spinal cord of amammal in response to first and second models of neuropathic or centralsensitization pain.
 17. A non-human animal according to claim 16,wherein said gene sequence becomes up regulated both in response tostreptozocin induced diabetes and in response to chronic constrictioninjury.
 18. A non-human animal according to claim 17, wherein theintroduced gene sequence encodes a kinase, a phosphatase, aphosphodiesterase, an ion channel protein, a DNA binding protein, anoxidoreductase or a hydrolase.
 19. A non-human animal according to claim16 which is C. elegans.
 20. A kit comprising; (a) affinity peptideand/or ligand and/or substrate for an expression product of a genesequence that is up-regulated in the spinal cord of a mammal in responseto a mechanistically distinct first and second models of neuropathic orcentral sensitization pain; and (b) a defined quantity of an expressionproduct of a gene sequence that is up-regulated in the spinal cord of amammal both in response to first and second models of neuropathic orcentral sensitization pain, for simultaneous, separate or sequential usein detecting and/or quantifying up-regulation of a gene sequence in thespinal cord of a mammal in response to first and second models ofneuropathic or central sensitization pain.
 21. A kit according to claim20, wherein the gene sequence encodes a kinase, a phosphatase, aphosphodiesterase, an ion channel protein, a DNA binding protein, anoxido reductase or a hydrolase.
 22. A kit comprising: (a) nucleic acidsequences capable of hybridization to a nucleic acid sequence that isup-regulated in the spinal cord of a mammal in response to first andsecond models of neuropathic or central sensitization pain; and (b) adefined quantity of one or more nucleic acid sequences capable ofhybridization to a nucleic acid sequence that is up-regulated in thespinal cord of a mammal in response to first and second models ofneuropathic or central sensitization pain, for simultaneous, separate orsequential use in detecting and/or quantifying up-regulation of a genesequence in the spinal cord of a mammal in response to first and secondmodels of neuropathic or central sensitization pain.
 23. A kit of claim22, wherein the gene sequence encodes a kinase, a phosphatase, aphosphodiesterase, an ion channel protein, a DNA binding protein, anoxido reductase or a hydrolase.
 24. A compound that modulates the actionof an expression product of a gene sequence that is up-regulated in thespinal cord of a mammal in response to first and second models ofneuropathic or central sensitization pain. 25 A compound according toclaim 24 wherein the gene sequence is listed in Table
 1. 26. A compoundaccording to claim 24 wherein the nucleotide sequence encodes a kinase,a phosphatase, a phosphodiesterase, an ion channel protein, a DNAbinding protein, an oxidoreductase or a hydrolase.
 27. A compoundaccording to claim 24 for use as a medicament.
 28. A compound accordingto claim 24 for the treatment or diagnosis of pain.
 29. A pharmaceuticalcomposition comprising a compound according to claim 24 and apharmaceutically acceptable carrier or diluent.
 30. Use of a compoundaccording to claim 24 in the manufacture of a medicament for thetreatment or diagnosis of pain.
 31. Use of a compound according to claim24 in the manufacture of a medicament for the treatment or diagnosis ofchronic pain.
 32. A method of treatment of pain, which comprisesadministering to a patient an effective amount of a compound accordingto claim 24.